Hypokalemia alters sex hormone and gonadotropin levels:
evidence that FSH may be required for luteinization
Francisco
Tejada1,
Asunción
Cremades1,
Manuel
Avilés2,
Maria T.
Castells2, and
Rafael
Peñafiel3
Departments of 1 Pharmacology,
2 Cell Biology, and
3 Biochemistry and Molecular
Biology, Faculty of Medicine, University of Murcia, 30100 Murcia,
Spain
 |
ABSTRACT |
Hypokalemia
produced different effects on steroid sex hormone concentrations in
plasma and ovary in the mouse. Estradiol levels were slightly
increased, whereas circulating progesterone was markedly decreased in
all estrous periods. The preovulatory surge of gonadotropins and the
secondary surge of follicle-stimulating hormone (FSH) at estrus were
also decreased, but basal levels of both gonadotropins were unaffected.
Supplementation with luteinizing hormone (LH), FSH, or
gonadotropin-releasing hormone (GnRH) at proestrus rapidly normalized
plasma and ovarian progesterone levels at this stage of the estrous
cycle. Plasma progesterone levels at diestrus were restored only by
combined treatment, at the periovulatory stage, with LH and FSH or GnRH
but not by LH or FSH alone. The results demonstrate a lack of
steroidogenic activity in the corpus luteum of the potassium-deficient
mice and, furthermore, that FSH plays an important role in
luteinization in the hypokalemic mice. We conclude that alteration of
the transcellular potassium gradient may affect the regulation of the
periovulatory surge of gonadotropins and progesterone secretion,
probably by altering the release of GnRH from the hypothalamus. In
addition, the results suggest that FSH may play a certain role as a
luteotropic hormone in mice.
potassium; corpus luteum; gonadotropin-releasing hormone; mice
 |
INTRODUCTION |
POTASSIUM is the most abundant intracellular cation in
mammalian cells, where it reaches a concentration of ~150 mM, which is higher than the concentration in plasma (4-5 mM). Although the
biochemical functions of potassium are not fully understood, the
existence of a potassium gradient between the internal and external
faces of the plasma membrane seems to be an important fact in the
maintenance of excitatory and secretory properties of neuronal and
secretory cells (15, 17, 31).
We recently found that potassium deficiency produces a marked decrease
in the circulating levels of testosterone (50). The reason for the fall
in testosterone production was related to the decrease in luteinizing
hormone (LH) secretion and was caused by alteration of the pulsatile
release of gonadotropin-releasing hormone (GnRH) from the hypothalamus
(49). These results showed that plasma potassium concentration is an
important factor in regulating androgenic status and fertility in male mice.
At present, it is not known whether the alteration of plasma potassium
concentration can affect hormonal and reproductive status in females.
Although it is well established in female mice that the synthesis and
release of ovarian sexual steroid hormones are regulated by
gonadotropins (20), it is also clear that, in female rodents, the
levels of plasma sexual steroid hormones exhibit periodical changes
related to the estrous cycle (6, 52). In contrast to the testes (25),
several cell types in the ovary participate in the synthesis and
release of steroids. The ability of either follicular cells, such as
theca and granulosa cells or interstitial cells, to produce steroids
depends on follicular development (23).
The pituitary gonadotropins LH and follicle-stimulating hormone (FSH),
which regulate steroidogenesis, maturation of ovarian follicles, and
luteinization, are secreted by the same gonadotropes (55). The
synthesis and release of both LH and FSH are stimulated by GnRH, which
is synthesized by hypothalamic neurons (8, 40, 53). GnRH
binds specifically to a seven-transmembrane-domain G protein-coupled
receptor that activates a number of intracellular secondary messengers,
including calcium, inositol phosphates, protein kinase C, and
mitogen-activated protein kinases (28). The mechanisms underlying the
pulsatile pattern of GnRH secretion are not well defined (33), although
several lines of evidence suggest that
Ca2+ is the intracellular
messenger that regulates GnRH exocytosis by GnRH neurons (32, 59). In
rodents, the secretory response of gonadotropes to GnRH varies during
the estrous cycle (42, 52): the release of GnRH is influenced by
steroid hormones (19, 39) and by environmental factors such as
light-day length, temperature, and food availability (13).
The steroidogenic actions of LH and FSH on ovarian cells are mediated
by cAMP via cAMP-dependent protein kinases (9), leading to the
phosphorylation of key substrates, including specific transcription (45). Key steps in the steroidogenic pathway are the inductions of
cytochrome P-450 cholesterol
side-chain cleavage enzyme and aromatase enzyme (20). Moreover, other
signaling transduction pathways, including protein kinase C and protein
tyrosine kinase as well as protein phosphatases, have also been
implicated in the steroidogenic gonadotropin function in the ovary (1,
21, 64). The role of gonadotropins in biosynthetic steroidogenic activity changes during the estrous cycle (20). Under the influence of
basal levels of FSH and LH, the ovary secretes estrogens, and after the
preovulatory surge of gonadotropins, the granulosa cells of the corpus
luteum of the ovary secrete large amounts of progesterone (20, 38).
Moreover, gonadotropins also participate in other ovarian functions;
FSH mainly stimulates the growth and maturation of the ovarian
follicle, whereas LH causes the follicular rupture and luteinization
(44, 54).
In the present study we have evaluated the effects produced by
potassium deficiency on the cyclical values of two steroid hormones,
estradiol and progesterone, in plasma and in ovary. We have also
studied the effect of hypokalemia on gonadotropin secretion throughout
the estrous cycle and the implication of gonadotropin alterations in
the normal development of corpus luteum.
 |
MATERIALS AND METHODS |
Animals and treatments.
Adult Swiss CD1 female mice were used in these experiments. Control
animals were fed standard chow (UAR A03, Panlab, Barcelona, Spain)
containing 7.5 g/kg potassium and water ad libitum. Potassium deficiency was produced by giving the mice a diet similar to the control diet but containing 120 mg/kg potassium (UAR 212K, Panlab) for
a period of 14-20 days. Potassium replenishment was achieved by
supplementation of the low-potassium diet with 1% KCl solution for 7 days instead of drinking water after 20 days of potassium deficiency.
The body weight of mice fed with the potassium-deficient diet decreased
slightly during treatment. Animals were maintained at 22°C ambient
temperature and 50% relative humidity under a controlled 12:12-h
light-dark cycle. Estrous cycles were monitored by daily vaginal
smears, and only mice exhibiting at least two consecutive 4-day estrous
cycles were included in the study. For collecting ovulated eggs,
oviducts were removed and placed in the well of a dish containing
phosphate buffer (pH 7.4). To collect oocyte-cumulus complexes, the
ampullary region of the oviduct was identified and torn open with fine
steel tweezers under a dissecting microscope (5). The oocyte-cumulus
complexes were isolated, and the cumulus cells were removed by
digestion for 5 min with sheep testicular hyaluronidase (1 mg/ml) (41).
The oocytes were identified and counted. For fertility evaluation, one
adult male of proven fertility and one control or potassium-deficient female were caged together for different periods of time. Females were
observed for signs of pregnancy, such as body weight increase and
delivery of fetuses by cesarian section at day
18 after a vaginal plug was observed.
Blood samples were collected under light ether anesthesia by cardiac
puncture, at 0900 and 1830 at proestrus or at 0900 at estrus and
diestrus (one puncture per animal). Plasma was obtained by
centrifugation at 4°C and was kept frozen at
70°C until
analysis. Mice were killed by decapitation under ether anesthesia, and
the ovaries were quickly removed and weighed. For histological
examination, the ovaries were fixed in 2% glutaraldehyde in 0.1 M
cacodylate buffer. Hypophyses were removed, fixed in 10% Formalin in
PBS at pH 7.2 for 7 h, and processsed for immunocytochemistry.
To determine progesterone turnover, the potent steroidogenic inhibitor
aminoglutethimide was used (10, 37). Control and potassium-deficient
mice were treated with aminoglutethimide (150 mg/kg ip, dissolved in
30% propylene glycol) and LH (from ovine pituitaries, 6 IU im,
dissolved in physiological saline) by following different schedules.
One group of control and potassium-deficient mice received only
aminoglutethimide, and animals were killed at periods of time after the
injection. Another group of potassium-deficient mice was treated
simultaneously with aminoglutethimide and LH, and samples were
collected at periods of time from 5 to 30 min.
For hormone replacement experiments, potassium-deficient mice were
treated with hypothalamic or hypophysial hormones by following different schedules. In one group (acute treatment),
potassium-deficient mice were treated with a single injection of LH (6 IU), FSH (6 IU), human chorionic gonadotropin (hCG, 6 IU), or GnRH (8 µg) at 0900 of proestrus and were killed 60 min after the injection. In another group (combination or repeated treatment), mice fed a
low-potassium diet for 15 days were treated as follows:
1) LH (6 IU) at proestrus and
estrus; 2) FSH (6 IU) at proestrus
and estrus; 3) LH (6 IU) at
proestrus; 4) LH (6 IU) and FSH
(6 IU) at proestrus and FSH (6 IU) at estrus;
5) LH (6 IU) at proestrus and FSH (6 IU) at estrus; 6) LH (6 IU) and FSH
(6 IU) at proestrus; and
7) three consecutive injections of
GnRH [8 µg each, 1 h apart, at the proestrus to mimic the surge
of gonadotropins (4)]. All hormones were administered by
intramuscular injection of the hormone dissolved in physiological
saline solution in the morning of proestrus and/or estrus.
Analytic methods.
Plasma potassium was analyzed using a potassium-selective electrode
(Beckman, Fullerton, CA).
Progesterone and estradiol were determined by ELISA with an Enzymun
Test kit supplied by Boehringer Mannheim Immunodiagnostics (Mannheim,
Germany). Concentrations of progesterone and estradiol in plasma were
measured in duplicate. Ovarian progesterone and estradiol
concentrations were determined after homogenization of ovaries with a
polytron (Brinkmann Instruments, Westbury, NY) in ice-cold ethanol
(1:20 wt/vol). The extract was centrifuged at 10,000 g for 20 min, the supernatant was
diluted in 50% ethanol containing 0.9% NaCl, and progesterone was
measured in duplicate. The intra-assay variation and sensitivity were
10% and 5 pg/tube, respectively.
The plasma LH and FSH concentrations were determined by RIA by use of
reagents supplied by the National Institute of Diabetes and Digestive
and Kidney Diseases (NIDDK, Bethesda, MD). Rat antigens rLH-I-9 and
rFSH-I-8 were iodinated with 125I
by the chloramine-T method (24) by following the recommendations supplied by the NIDDK. Rat LH antiserum (rabbit) NIDDK anti-rLH-S-11 and rat FSH antiserum (rabbit) NIDDK anti-rFSH-S-11 were used, and the
antigen-antibody complexes were precipitated with protein A-Sepharose
(Sigma Chemical, St Louis, MO). LH and FSH concentrations are expressed
using rLH-RP-3 and rFSH-RP-2, respectively, as standards. The
intra-assay variation and sensitivity were 7% and 3.5 pg/tube for LH
and 8% and 2.5 pg/tube for FSH.
Ornithine decarboxylase activity was determined in the cytosol of
ovarian extracts (12,000 g
supernatant) by measuring the release of
14CO2
from
L-[1-14C]ornithine
according to a previously described protocol (49).
Histology and immunocytochemistry.
Ovaries from the different experimental groups were removed by
dissection and fixed for 2 h at 4°C in 2% glutaraldehyde in 0.1 M
cacodylate buffer (pH 7.4). After washing, the samples were postfixed
with osmium tetraoxide and routinely processed in Epon 812 resin.
Semi-thin sections (1 µm thick) obtained using glass knives on an LKB
ultramicrotome were stained with toluidine blue.
Hypophyses from each experimental group
(n = 5) were fixed in 10% Formalin in
PBS pH 7.2 for 7 h. After washing in PBS, tissues were processed and
embedded in paraffin. Five-microgram sections were obtained and
collected on slides coated with
poly-L-lysine. Sections were
immunostained according to the peroxidase/antiperoxidase (PAP) method
(58) with antibodies against mouse LH and mouse FSH (1:250; Biogenesis,
Poole, England). Sections from each experimental group were stained
under the same conditions. The antibody was substituted by PBS in
control sections. The intensity of immunoreaction was evaluated using
an Imco 10 Image computer (Kontron bildanalyse, Eching, Germany) and
MIP software (Microm, Barcelona, Spain). All microscopic images were
captured in one session with the same illumination conditions by means
of a video camera. The digital images consisted of 512 × 512 pixels, where each pixel showed a number between 0 (dark) and 255 (bright) indicating the intensity of transmitted light or gray level at
that point. Measurement of the gray level showed the intensity of
immunoreaction, the higher gray level indicating a lower
immunoreaction. One hundred cells for each experimental group were
evaluated. Cytoplasm from reactive cells was selected interactively,
and mean gray level per cell was counted. Median value and standard
deviation were calculated.
Statistics.
Results are given as means ± SD. Statistical comparisons were
calculated by ANOVA followed by the post hoc Newman-Keuls multiple range test with a Prism program (GraphPad Software, San Diego, CA).
P < 0.05 was considered
statistically significant. LH levels at proestrus were assessed by
cluster analysis.
 |
RESULTS |
Table 1 shows that potassium deficiency
produced by feeding adult female mice with a potassium-depleted diet
caused a clear decline in plasma potassium concentration and that the
circulating levels of this cation were restored when the diet of
potassium-deficient animals was supplemented with KCl. Potassium
deficiency produced a significant decrease in ovary weight. The
reproductive capacity of the female mice was dramatically reduced in
the potassium-deficient animals. None of the potassium-deficient
animals became pregnant in contrast to control female mice. In the
potassium-deficient animals (Table 1), the estrous cycle was normal,
but the number of oocytes per oviduct was reduced.
Plasma estradiol and progesterone values in control adult Swiss female
mice exhibited clear periodical changes during the estrous cycle,
similar to those found in other rodents (52). Potassium deficiency
produced a slight increase in the circulating levels of estradiol at
all of the estrous periods studied, except in diestrus II (Fig.
1A).
However, in potassium-deficient mice, plasma progesterone
concentrations were dramatically reduced in all estrous stages (Fig.
1B). Progesterone values were
normalized after potassium replenishment to control values.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of potassium deficiency on plasma estradiol
(A) and progesterone
(B) concentrations at different
stages of estrous cycle in female mice. Animals were fed with a
potassium-deficient diet for 20 days. Results are means ± SD for nos.
of animals in parentheses.
a P < 0.001 vs. K+ deficient.
|
|
Table 2 shows progesterone and estradiol
values in ovarian tissue in different stages of the estrous cycle in
control and potassium-deficient mice. It can be seen that potassium
deficiency produced a considerable decrease in the progesterone
content, ranging from 22% in proestrus to 45% in estrus. Potassium
replenishment restored progesterone concentrations to normal values.
Conversely, the estradiol content in the ovary was significantly
increased (10-27%) by potassium deficiency. The comparison of
plasma and ovarian concentration of progesterone in control and
potassium-deficient mice shows that a moderate decrease in ovarian
progesterone is associated with a drastic fall in the circulating
concentrations of this steroid. This suggests that potassium deficiency
does not affect the synthesis and secretion of progesterone equally. That potassium-deficient mice with an ovarian progesterone content >50% above basal levels are unable to maintain a normal pattern of
plasma progesterone can be interpreted by assuming that a part of
ovarian progesterone content may be present in a nonsecretable pool. To
determine whether this progesterone was in a metabolically active form,
the turnover of this steroid in ovarian cells was determined in both
control and potassium-deficient mice by using aminoglutethimide, a
potent inhibitor of steroidogenesis (10, 37). Figure
2 shows that, in control females,
progesterone decay in plasma and ovary was similar, presenting a
half-time of ~8-10 min. In potassium-deficient mice, the
turnover of progesterone in the ovary was similar to that of controls,
indicating that, in this case, progesterone is metabolized but not
secreted into the plasma. However, when LH was coadministered with
aminoglutethimide, ovarian progesterone continuously decreased, whereas
plasma progesterone showed a transient rise with a maximum of ~8
ng/ml 10 min after the administration (Fig.
3), which indicates that ovarian
progesterone from potassium-deficient mice can be secreted after
stimulation by exogenous LH.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Progesterone turnover in control and potassium-deficient female mice.
Progesterone was determined in plasma and ovaries of control and
potassium-deficient mice at different times after aminoglutethimide
(150 mg/kg ip) injection. Results are means obtained from 3 animals/point.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of luteinizing hormone (LH) on plasma and ovarian progesterone
levels in potassium-deficient mice treated with aminoglutethimide.
Potassium-deficient female mice (diestrus II) were treated with
aminoglutethimide (150 mg/kg ip) and LH (6 IU im), and progesterone
concentrations were measured at different times after injection. Each
point is the mean of duplicate determinations from 3 mice. SD were
<10% of means.
|
|
To know whether hypokalemia directly affects the ovarian ability to
produce and release progesterone or whether it alters pituitary
function, potassium-deficient mice were injected with an intramuscular
injection of gonadotropins, and plasma and ovarian progesterone were
determined after 60 min. This treatment restored progesterone levels in
both the plasma and the ovary (Table 3), which suggests that the progesterone changes in potassium-deficient mice may be related to alteration in the release of gonadotropins from
the pituitary. The administration of GnRH, which can evoke a peak of LH
and FSH, also restored progesterone levels (Table 3), suggesting that
potassium deficiency may influence the hypothalamic release of GnRH.
Because GnRH is involved in both synthesis and release of gonadotropins
by the gonadotropes (13), we compared plasma LH and FSH concentrations
and immunoreactive pituitary LH and FSH levels in control and
potassium-deficient mice. Immunoreaction intensity of the cytoplasm of
reactive gonadotropes was slightly higher in the controls than in the
potassium-deficient mice for both LH and FSH (Table
4).
View this table:
[in this window]
[in a new window]
|
Table 3.
Effects of hCG, LH, FSH, and GnRH on progesterone levels of
potassium-deficient female mice at proestrus
|
|
Figure 4,
A and
B, shows the values of gonadotropins
in the plasma of control and potassium-deficient mice at different
stages of the estrous cycle. It can be seen that hypokalemia does not affect LH values during estrus and diestrus, but it decreases the LH
surge at proestrus (P < 0.01 by
cluster analysis). In the case of FSH, both the proestrus and the early
estrus surges were significantly decreased by potassium deficiency,
whereas the low value at diestrus was not affected. Replenishment with
KCl restored gonadotropin values. LH values at proestrus varied
markedly, and potassium-deficient mice do not reach the high values
found in control animals. The analysis of frequencies of mice with LH
values at proestrus over a fixed LH concentration in the three
experimental groups showed significant differences between the
potassium-deficient mice and the other groups (Fig.
5). Ovarian activity at 2300 of proestrus
of ornithine decarboxylase, an enzyme induced by the LH surge at
proestrus (63), was dramatically decreased in potassium-deficient mice
(1.82 ± 0.93 vs. 23.6 ± 8.2 nmol
14CO2 · h
1 · g
1
in the control group), supporting a decrease in the LH surge in the
potassium-deficient mice at proestrus. The decrease of both
gonadotropins at proestrus (Fig. 4, A
and B), together with the fall of
progesterone during the diestrus (Fig. 1), suggests that hypokalemia
may alter the development of the corpus luteum as a result of decreased
gonadotropin secretion. Histological examination of the ovaries of the
different groups did not show significant differences in the size of
the corpora lutea contained in the ovary. However, the morphology of
the granulosa lutein cells of the corpus luteum varied according to the
experimental group considered. Thus the cytoplasm of granulosa lutein
cells of control mice was essentially devoid of granules (Fig.
6A). However, in the potassium-deficient group, abundant granules were observed in the cytoplasm (Fig. 6B).
In the group of potassium deficiency supplemented with LH and FSH at
the periovulatory period, the granulosa lutein cells showed a
morphology similar to that of the control group (Fig.
6C). Because plasma progesterone
levels at diestrus are mainly secreted by the corpus luteum, the fall in the levels of this hormone in the potassium-deficient mice at this
stage suggests that the biochemical changes related to the
steroidogenic capacity of this gland may not have been produced.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of potassium deficiency on LH
(A) and follicle-stimulating hormone
(FSH, B) values at different stages
of estrous cycle in female mice. Results are means ± SD for nos. of
animals in parentheses.
a P < 0.001 vs. K+ deficient;
b P < 0.01 vs. K+ deficient.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Frequency distribution of animals with plasma LH concentrations at
proestrus above different fixed LH values, expressed by use of rLH-RP-3
as standard. Nos. of animals are as in Fig. 3.
* P < 0.05 by ANOVA test for
values >5 ng/ml LH.
|
|

View larger version (81K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of potassium deficiency on granulosa lutein cells of mouse
ovary. A: control animals; cytoplasm
of granulosa lutein cells is devoid of granules.
B: potassium-deficient mice; abundant
granules are detected in cytoplasm of granulosa lutein cells (arrows);
inset, a granulosa lutein cell at high
magnification. C: potassium-deficient
animals supplemented with FSH and LH at proestrus and FSH at estrus; no
cytoplasmic granules are observed in granulosa lutein cells. Bar, 24.7 µm (A and
C); 15 µm
(B); 12 µm,
inset.
|
|
To know the possible relationship between the inability of the corpus
luteum to secrete steroids in potassium-deficient mice and alteration
of gonadotropin secretion, we examined the role of each gonadotropin in
luteinization by administering exogenous LH, FSH, and GnRH at specific
times of the estrus cycle, mimicking the normal pattern of plasma
gonadotropins. Table 5 shows that LH or FSH
alone, given at proestrus or at proestrus and estrus, was unable to
restore the secretory capacity of progesterone by the corpus luteum at
diestrus. Conversely, administration of both gonadotropins by following
different schedules completely restored plasma and ovarian progesterone
at diestrus. The sustained administration of GnRH at proestrus also
normalized the secretory capacity of the corpus luteum at diestrus.
Prolactin injection (10 IU) or sulpyride treatment (30 mg/kg) at
proestrus and estrus did not increase progesterone concentration at
diestrus (results not shown).
View this table:
[in this window]
[in a new window]
|
Table 5.
Effect of LH, FSH, and GnRH on secretion of progesterone by corpus
luteum of potassium-deficient female mice
|
|
 |
DISCUSSION |
Our results show that potassium deficiency produces different effects
on ovarian and plasma steroid hormones in female mice and suggest that
normal levels of potassium are essential for the maintenance of correct
regulatory control of steroid sex hormones.
That potassium deficiency decreases plasma progesterone while slightly
increasing circulating estrogens indicates that the response of ovarian
hormones to hypokalemia is specific and correlates with the changes
observed in the reproductive physiology of female mice. These results
complement our previous findings showing that potassium deprivation
produced a dramatic decrease in circulating testosterone and in the
activity of enzymes induced by androgens in male mice (50). Taken
together, our results clearly show that hypokalemia causes infertility
in both male and female mice.
The absence of augmented plasma progesterone levels during proestrus in
the potassium-deficient animals also suggests that the rise of this
steroid hormone is not essential for the ovulatory process or for the
change in vaginal epithelium throughout the estrous cycle. The
persistence of essentially normal estradiol levels at different stages
of the estrous cycle in potassium-deficient mice, together with the
apparently normal pattern of the vaginal smear along the estrous cycle,
also supports the postulated role of this hormone in the proliferation
of vaginal epithelium (22).
Interestingly, our results show that there is no correlation between
plasma progesterone concentration and ovarian progesterone content,
because the dramatic decrease in plasma progesterone observed in the
potassium-deficient mice is not accompanied by a proportional decrease
in ovarian progesterone. This observation cannot be attributed to
changes in ovarian progesterone turnover, because the half-life of
progesterone was similar in control and potassium-deficient mice. The
rise in plasma progesterone levels found after treatment of
potassium-deficient mice with aminoglutethimide and LH indicates that
ovarian progesterone in the preovulatory follicle of
potassium-deficient mice can be secreted after an adequate surge of LH.
This finding demonstrates that synthesis and secretion of progesterone
by ovarian structures do not always take place simultaneously.
Moreover, this finding is in agreement with a recent suggestion that
plasma levels of progesterone are not a precise indicator of follicular
levels of steroids, supporting an autocrine role for progesterone in
follicular rupture (62).
The decrease in plasma and ovarian progesterone induced by hypokalemia
seems to be due to a reduction in the secretion of gonadotropins,
probably as a consequence of alteration in the release of GnRH by the
hypothalamus. This finding corroborates and completes our recent study,
which showed that in potassium-deficient male mice, the alteration of
GnRH release was associated with decreased secretion of LH from the
pituitary (49). Although in both male and female, gonadotropin
secretion is under control of hypothalamic GnRH, the secretory pattern
of GnRH and gonadotropins in the female mice is complex and varies
during the estrous cycle (42). In this regard, it is well established
that the secretion of LH and FSH by the pituitary is under control of
several hormones and ovarian factors (7, 13, 34, 60) that determine a differential release of each gonadotropin along the estrous cycle.
Our results showed that LH and FSH surges at proestrus and the
secondary surge of FSH at estrus were altered in the
potassium-deficient mice. The fall in the preovulatory surge of LH and
FSH, associated with the high level of estrogens in the morning of
proestrus, suggests that the release of GnRH by the hypothalamus may be
altered by hypokalemia, indicating that the positive feedback of
estradiol on GnRH liberation (12, 35) is blocked by hypokalemia. The analysis of LH and FSH immunoreactivity in the pituitary at proestrus suggests that the fall of GnRH release may affect the secretion more
than the synthesis of gonadotropins. The marked decrease of FSH in
estrus may be related to the diminution of LH and progesterone at
proestrus, because it is well established that the secretion of FSH in
estrus depends on both the primary surge of gonadotropins (47, 57) and
the permissive absence of circulating inhibin (2, 47, 65).
It is widely accepted that the preovulatory surge of gonadotropins is
implicated in the release of the ovum and in the transformation of the
follicle into the corpus luteum (18, 46). Although a simultaneous surge
of both LH and FSH precedes follicular rupture in most mammals, it is
the larger surge of LH that is felt to be essential for ovulation and
luteinization (11, 29, 56). Our results show that the values of LH and
FSH found at proestrus in the potassium-deficient mice, although
considerably lower than those found in the control group, are
sufficient to promote the follicular rupture. However, they are clearly
not sufficient for the formation of a functional corpus luteum, as
demonstrated by the dramatic diminution of progesterone secretion at
diestrus and altered morphology. This indicates that the levels of
gonadotropins required for ovulation differ from those necessary for
the complete development of the corpus luteum. This view is not in
agreement with the earlier statement that the preovulatory surges of
both LH and FSH may represent a protective mechanism to ensure an
optimal ovulatory stimulus (16), because both gonadotropins are
required for the development of a functional corpus luteum. Our model
demonstrates that the infertility associated with potassium deficiency
may be attributable to the alteration of the luteinizing process rather than to ovulation and suggests that luteinization may be more sensitive
than ovulation to the fall in gonadotropins. Although it seems clear
that potassium deficiency affects the preovulatory surge of
gonadotropins, it is difficult to conclude from the analysis of the
gonadotropin values whether their basal concentrations have been
modified by hypokalemia. However, the maintenance of plasma estradiol
values together with the existence of a normal ovulatory process
suggests that basal levels of gonadotropins are not seriously affected
by potassium deprivation. The fact that low levels of circulating
progesterone are compatible with normal basal levels of gonadotropins
also suggests that the negative feedback of progesterone on basal
gonadotropin secretion is irrelevant. Our data, however, support the
idea that the FSH surge in the periovulatory period may be required to
promote the synthesis and secretion of progesterone by the corpus
luteum. The decrease in the ovulatory rate can be explained by the
observed decrease in the secondary FSH surge, because it is known that
a sustained elevation of serum FSH is responsible for the recruitment
of the ensuing cohort of follicles (26). It has been generally
accepted, of course, that LH and prolactin are the two identifiable
luteotropic hormones in the rat (30, 44, 48); however, our study
suggests that, in mice, FSH may play an important role in the secretion of progesterone, the primary function of the corpus luteum. That neither exogenous prolactin administration nor hyperprolactinemia induced by the dopaminergic receptor blocker sulpyride was able to
induce luteinization in the potassium-deficient mice also supports the
thesis that, in our model, the fall in the periovulatory surge of both
LH and FSH is responsible for altered corpus luteum formation.
Our results show that the treatment of potassium-deficient mice with
exogenous GnRH normalizes progesterone secretion and indicate that
pituitary gonadotrophs remain responsive to this peptide even under
conditions of potassium deprivation. This suggests that plasma
progesterone reduction elicited by hypokalemia may be related to an
alteration in the mechanism of pulsatile release of GnRH by
hypothalamic GnRH-producing neurons (33). However, the possibility that
the response to physiological amounts of GnRH in the
potassium-deficient mice is decreased cannot be completely excluded.
Independently of the molecular mechanisms by which potassium may affect
the regulation of the hypothalamo-hypophysial-gonadal axis, our results
show that a moderate decrease in the plasma potassium concentration
seriously affects the reproductive capacity of mice. The changes
induced by potassium deficiency in the reproductive system of female
mice are readily reversible, because the maintenance of a functional
follicular development and ovulation under potassium-deprived conditions only requires rapid stimulation of progesterone secretion by
the corpus luteum after normalization of potassium supply. The
impairment of reproductive function produced by potassium deficiency in
mice may represent a protective mechanism against a condition such as
pregnancy, which requires a large amount of this essential ion. In this
regard, there are different pathological states and pharmacological
treatments (3, 27, 36, 51, 61) in humans associated with alteration in
potassium homeostasis that may be accompanied by other disturbances of
hormonal status and that may adversely affect reproductive functions.
In our opinion, this experimental system seems to offer a good model
for study of the role of sex hormones in the regulation of
hypothalamic-hypophysary function in intact animals.
In conclusion, our results indicating that potassium deficiency
severely affects the reproductive hormones, not only in male but also
in female mice, together with the reported effects of hypokalemia on
growth hormone, aldosterone, and insulin secretion (14, 61), suggest
that normal levels of potassium are required for maintenance of
reproductive and other endocrine functions in mammals.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Leonor Pinilla for advice in LH and FSH
determinations, Dr. Derek Smyth for critical reading of the manuscript, and the National Institute of Diabetes and Digestive and Kidney Diseases for the reagents for the LH and FSH RIA.
 |
FOOTNOTES |
This work was supported by grants from Fondo de Investigacion Sanitaria
(FIS 98/0503), Ministerio de Sanidad y Consumo, Spain, and the Seneca
Program, Comunidad Autonoma de Murcia, Spain.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: R. Peñafiel, Dept. of Biochemistry
and Molecular Biology, Faculty of Medicine, Univ. of Murcia, 30100 Murcia, Spain.
Received 10 March 1998; accepted in final form 13 August 1998.
 |
REFERENCES |
1.
Abayasekara, D. R. E.,
S. L. Ford,
S. J. Persaud,
and
P. M. Jones.
Role of phosphoprotein phosphatases in the corpus luteum. II. Control of progesterone secretion by isolated rat luteal cells.
J. Endocrinol.
150:
213-221,
1996[Abstract].
2.
Ackland, J. F.,
J. D'Agostino,
S. Ringstrom,
J. P. Hostetler,
B. Mann,
and
N. B. Schwartz.
Circulating radioimmunoassayable inhibin during period of transient follicle-stimulating hormone rise: secondary surge and unilateral ovariectomy.
Biol. Reprod.
43:
347-352,
1990[Abstract].
3.
Adrogue, H. J.,
E. D. Lederer,
W. N. Suki,
and
G. Ekonyan.
Determination of plasma potassium levels in diabetic ketoacidosis.
Medicine
65:
163-168,
1986[Medline].
4.
Aiyer, M. S.,
S. A. Chiappa,
and
G. Fink.
A priming effect of luteinizing hormone releasing factor on the anterior pituitary gland in female rat.
J. Endocrinol.
62:
573-588,
1974[Medline].
5.
Avilés, M.,
L. Jaber,
M. T. Castells,
F. W. K. Kan,
and
J. Ballesta.
Modifications of the lectin binding pattern in the zona pellucida of rat after fertilization.
Mol. Reprod. Dev.
44:
370-381,
1996[Medline].
6.
Butcher, R. L.,
W. E. Collins,
and
N. W. Fugo.
Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17
throughout the estrous cycle of the rat.
Endocrinology
94:
1704-1708,
1974[Medline].
7.
Byrne, B.,
P. A. Fowler,
and
A. Templeton.
Role of progesterone and nonsteroideal ovarian factors in regulating gonadotropin-releasing hormone self priming in vitro.
J. Clin. Endocrinol. Metab.
81:
1454-1459,
1996[Abstract].
8.
Conn, P. M.,
C. A. McArdle,
W. V. Andrews,
and
W. R. Huckle.
The molecular basis of gonadotropin-releasing hormone (GnRH) action in the pituitary gonadotrope.
Biol. Reprod.
36:
17-35,
1987[Medline].
9.
Darbon, J. M.,
J. Ursely,
and
P. Laymarie.
Stimulation by LH of cyclic AMP-dependent protein kinase activity in bovine corpus luteum slices.
FEBS Lett.
63:
159-163,
1976[Medline].
10.
Dexter, R. N.,
L. M. Fishman,
R. L. Ney,
and
G. W. Liddle.
Inhibition of adrenal corticosteroid by aminoglutethimide: studies on the mechanism of action.
J. Clin. Endocrinol.
27:
473-480,
1967[Medline].
11.
Ely, C. A.,
and
N. B. Schwartz.
Elucidation of the role of the luteinizing hormone in estrogen secretion and ovulation by use of antigonadotropic sera.
Endocrinology
89:
1103-1108,
1971[Medline].
12.
Fink, G.
Feedback actions of target hormones on the hypothalamus and pituitary with special reference to gonadal steroids.
Annu. Rev. Physiol.
41:
571-585,
1979[Medline].
13.
Fink, G.
Gonadotropin secretion and its control.
In: The Physiology of Reproduction, edited by E. Knobil,
and J. D. Neill. New York: Raven, 1988, p. 1349-1377.
14.
Flyvberg, A.,
I. Dorup,
M. E. Everts,
and
H. Orskov.
Evidence that potassium deficiency induces growth retardation through reduced growth hormone and insulin growth factor I production.
Metabolism
40:
769-775,
1991[Medline].
15.
Fregly, M. J.
Sodium and potassium.
Annu. Rev. Nutr.
1:
69-93,
1981[Medline].
16.
Galdway, A. B.,
P. S. Lapolt,
A. Tsafriri,
L. M. Dargan,
I. Boime,
and
A. J. W. Hsueh.
Recombinant follicle-stimulating hormone induces ovulation and tissue plasminogen activator expression in hypophysectomized rats.
Endocrinology
127:
3023-3028,
1990[Abstract].
17.
Giebisch, G.,
G. Mainic,
and
R. W. Berliner.
Control of renal potassium excretion.
In: The Kidney, edited by B. M. Brenner. Philadelphia: Saunders, 1996, p. 371-407.
18.
Goldman, B. D.,
and
V. B. Mahesh.
Fluctuations in pituitary FSH during the ovulatory cycle in the rat and a possible role of FSH in the induction of ovulation.
Endocrinology
83:
97-106,
1968[Medline].
19.
Goodman, R. L.
A quantitative analysis of the physiological role of estradiol and progesterone in the control of tonic and surge secretion of luteinizing hormone in the rat.
Endocrinology
102:
142-150,
1978[Medline].
20.
Gore-Langton, R. E.,
and
D. T. Armstrong.
Follicular steroidogenesis and its control.
In: The Physiology of Reproduction, edited by E. Knobil,
and J. D. Neill. New York: Raven, 1994, p. 571-628.
21.
Gornberg-Malool, S.,
R. Ziv,
V. Re'em,
I. Posner,
A. Levitzki,
and
J. Orly.
Tyrphostins inhibit follicle-stimulating hormone-mediated functions in cultured rat ovarian granulosa cells.
Endocrinology
132:
362-370,
1993[Abstract].
22.
Grasso, P.,
and
L. E. Reichert.
In vivo effects of follicle-stimulating hormone related synthetic peptides on the mouse estrous cycle.
Endocrinology
137:
5370-5375,
1996[Abstract].
23.
Greenwald, G. S.,
and
S. K. Roy.
Follicular development and its control.
In: The Physiology of Reproduction, edited by E. Knobil,
and J. D. Neill. New York: Raven, 1994, p. 629-723.
24.
Greenwood, F. C.,
W. M. Hunter,
and
J. S. Glover.
The preparation of 131I-labeled human growth hormone of high specific radiactivity.
Biochem. J.
89:
114-123,
1963.
25.
Hall, P. F.
Testicular steroid synthesis: organization and regulation.
In: The Physiology of Reproduction, edited by E. Knobil,
and J. D. Neill. New York: Raven, 1988, p. 975-985.
26.
Hoak, D. C.,
and
N. B. Schwartz.
Blockade of recruitment of ovarian follicles by suppression of the secondary surge of follicle-stimulating hormone with porcine follicular fluid.
Proc. Natl. Acad. Sci. USA
77:
4953-4956,
1980[Abstract].
27.
Hood, V. L.
Fluid and electrolyte disturbances during starvation.
In: Fluids and Electrolytes, edited by J. P. Kokko,
and R. L. Tannen. Philadelphia: Saunders, 1986, p. 712-732.
28.
Kaiser, U. B.,
P. C. Conn,
and
W. W. Chin.
Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor-expressing pituitary cell lines.
Endocr. Rev.
18:
46-70,
1997[Abstract/Free Full Text].
29.
Kelly, W. A.,
and
H. A. Robertson.
The suppression of ovulation in the rat by rabbit anti-ovine LH serum.
J. Endocrinol.
27:
127-128,
1963.
30.
Keyes, P. I.,
and
M. C. Wiltbank.
Endocrine regulation of the corpus luteum.
Annu. Rev. Physiol.
50:
465-482,
1988[Medline].
31.
Kolb, M. A.
Potassium channels in excitable and non-excitable cells.
Rev. Physiol. Biochem. Pharmacol.
115:
51-91,
1993.
32.
Krsmanovic, L. Z.,
S. S. Stojilkovic,
F. Merelli,
S. M. Dufour,
M. A. Virmani,
and
K. J. Catt.
Calcium signalling and episodic secretion of gonadotropin releasing hormone in hypothalamic neurons.
Proc. Natl. Acad. Sci. USA
89:
8462-8466,
1992[Abstract].
33.
Krsmanovic, L. Z.,
S. S. Stojilkovic,
and
K. J. Catt.
Pulsatile gonadotropin-releasing hormone release and its control.
Trends Endocrinol. Metab.
7:
56-59,
1996.
34.
Leaden, C. A.,
and
S. P. Kalra.
Stimulation with estrogen and progesterone of luteinizing hormone (LH) releasing hormone release from perifused adult female rat hypothalami: correlation with the LH surge.
Endocrinology
114:
51-56,
1984[Abstract].
35.
Legan, S. J.,
and
F. J. Karsch.
A daily signal for the LH surge in the rat.
Endocrinology
96:
57-62,
1975[Abstract].
36.
Lindinger, M. I.
Potassium regulation during exercise and recovery in humans: implications for skeletal and cardiac muscle.
J. Mol. Cell. Cardiol.
27:
1011-1012,
1995[Medline].
37.
Lipner, H.,
and
R. O. Greep.
Inhibition of steroidogenesis at various sites in the biosynthetic pathway in relation to induced ovulation.
Endocrinology
88:
602-607,
1971[Medline].
38.
Lostroh, A. J.,
and
R. E. Johnson.
Amounts of interstitial cell-stimulating hormone and follicle-stimulating hormone required for follicular development, uterine growth and ovulation in the hypophysectomized rat.
Endocrinology
79:
991-996,
1966[Medline].
39.
McCann, S. M.
Effect of progesterone on plasma luteinizing hormone activity.
Am. J. Physiol.
202:
601-604,
1962.
40.
Meites, J.,
and
W. E. Somtag.
Hypothalamic hypophysiotropic hormones and neurotransmitter regulation: current views.
Annu. Rev. Pharmacol. Toxicol.
21:
295-322,
1981[Medline].
41.
Miller, D. J.,
X. Gong,
G. Decker,
and
B. D. Shur.
Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.
J. Cell Biol.
123:
1431-1440,
1994[Abstract].
42.
Murr, S. M.,
I. I. Geschwind,
and
G. E. Bradford.
Plasma LH and FSH during different oestrous cycle conditions in mice.
J. Reprod. Fert.
32:
221-230,
1973[Medline].
43.
Niswender, G. D.,
and
T. M. Nett.
The corpus luteum and its control in infraprimate species.
In: The Physiology of Reproduction, edited by E. Knobil,
and J. D. Neill. New York: Raven, 1994, p. 781-816.
44.
Niswender, G. D.,
H. R. Sawyer,
T. T. Chen,
and
D. B. Endras.
Action of luteinizing hormone at the luteal cell level.
In: Advances in Sex Hormone Research,, edited by J. A. Thomas,
and R. L. Singhal. Baltimore, MD: Urban and Schwarzenberg, 1980, p. 153-185.
45.
Richards, J. S.
Hormonal control of gene expression in the ovary.
Endocr. Rev.
15:
725-751,
1994[Medline].
46.
Richards, J. S.,
and
L. Hedin.
Molecular aspects of hormone action in ovarian follicular development, ovulation and luteinization.
Annu. Rev. Physiol.
50:
441-463,
1988[Medline].
47.
Rivier, C.,
V. Roberts,
and
W. Vale.
Possible role of luteinizing hormone and follicle-stimulating hormone in modulating inhibin secretion and expression during the estrous cycle.
Endocrinology
125:
876-882,
1989[Abstract].
48.
Rothchild, I.
The regulation of the mammalian corpus luteum.
Rec. Prog. Horm. Res.
37:
183-283,
1981[Medline].
49.
Sánchez-Capelo, A.,
M. T. Castells,
A. Cremades,
and
R. Peñafiel.
Hypokalemia decreases testosterone production in male mice by altering luteinizing hormone secretion.
Endocrinology
137:
3738-3743,
1996[Abstract].
50.
Sánchez-Capelo, A.,
A. Cremades,
F. Tejada,
T. Fuentes,
and
R. Peñafiel.
Potassium regulates plasma testosterone and renal ornithine decarboxylase in mice.
FEBS Lett.
333:
32-34,
1993[Medline].
51.
Sánchez-Capelo, A.,
F. Tejada,
M. T. Castells,
F. Monserrat,
A. Cremades,
and
R. Peñafiel.
Influence of potassium homeostasis on testosterone levels and renal androgenic effects.
In: Metal Ions in Biology and Medicine, edited by P. Collery,
J. Corbella,
J. L. Domingo,
J. E. Etienne,
and J. M. Llobet. Paris: John Libbey Eurotext, 1996, p. 429-432.
52.
Savoy-Moore, R. T.,
N. B. Schwartz,
S. A. Ducan,
and
J. C. Marshall.
Pituitary gonadotropin-releasing hormone receptors during the rat estrous cycle.
Science
209:
942-944,
1980[Medline].
53.
Schally, A. V.,
A. Arimuta,
and
A. J. Kastin.
Hypothalamic regulatory hormones.
Science
173:
341-350,
1973.
54.
Schwartz, N. B.
The role of FSH and LH and their antibodies on follicle growth and on ovulation.
Biol. Reprod.
10:
263-272,
1974.
55.
Schwartz, N. B.
Follicle stimulating hormone and luteinizing hormone: a tale of two gonadotropins.
Can. J. Physiol. Pharmacol.
73:
675-684,
1995[Medline].
56.
Schwartz, N. B.,
and
J. J. Gold.
Effect of a single dose of anti-LH serum at proestrus on the rat estrous cycle.
Anat. Rec.
157:
137-149,
1967[Medline].
57.
Schwartz, N. B.,
and
W. L. Talley.
Effects of exogenous LH or FSH on endogenous FSH, progesterone and estradiol secretion.
Biol. Reprod.
17:
820-828,
1978.
58.
Stenberger, L. A.
The unlabeled antibody peroxidase-antiperoxidase (PAP) method.
In: Immunocytochemistry in Basic and Clinical Immunology, edited by S. Cohen,
and R. T. McCluskey. New York: Wiley, 1979, p. 104-169.
59.
Stojilkovic, S. S.,
L. Z. Krsmanovic,
D. J. Spergel,
M. Tomic,
and
K. J. Catt.
Calcium signalling and episodic secretory responses of GnRH neurons.
Methods Neurosci.
20:
68-84,
1994.
60.
Suter, D. E.,
and
N. B. Schwartz.
Effects of glucocorticoids on secretion of luteinizing hormone and follicle-stimulating hormone by female rat pituitary cells in vitro.
Endocrinology
117:
849-854,
1985[Abstract].
61.
Tannen, R. L.
Disorders of potassium balance.
In: The Kidney, edited by B. M. Brenner,
and F. C. Rector. Philadelphia: Saunders, 1991, p. 805-859.
62.
Tsafriri, A.,
and
N. Dekel.
Molecular mechanisms in ovulation.
In: Molecular Biology of the Female Reproductive System, edited by J. K. Findlay. San Diego, CA: Academic, 1994, p. 207-258.
63.
Williams-Ashman, H. G.,
J. Jänne,
G. L. Coppoc,
M. E. Geroch,
and
A. Schenone.
New aspects of polyamine biosynthesis in eukariotic organisms.
Adv. Enzyme Regul.
10:
225-245,
1972[Medline].
64.
Wiltbank, M. C.,
M. G. Diskin,
J. C. Flores,
and
G. D. Niswender.
Regulation of the corpus luteum by protein kinase C. II. Inhibition of lipoprotein-stimulated steroidogenesis by prostaglandin F2
.
Biol. Reprod.
4:
239-245,
1990[Medline].
65.
Woodruff, T. K.,
J. D'Agostino,
N. B. Schwartz,
and
K. E. Mayo.
Decreased inhibin gene expression in preovulatory follicles requires primary gonadotropin surges.
Endocrinology
124:
2193-2199,
1989[Abstract].
Am J Physiol Endocrinol Metab 275(6):E1037-E1045
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society