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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

                              
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Table 1.   Effect of potassium deficiency on plasma potassium, ovarian weight, and reproductive functions

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.


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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.

                              
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Table 2.   Effect of potassium deficiency on ovarian progesterone and estradiol


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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.


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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).

                              
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Table 3.   Effects of hCG, LH, FSH, and GnRH on progesterone levels of potassium-deficient female mice at proestrus

                              
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Table 4.   Effect of potassium deficiency on LH and FSH immunoreactive cells in female mouse adenohypophysis

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.


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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.


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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.


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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).

                              
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Table 5.   Effect of LH, FSH, and GnRH on secretion of progesterone by corpus luteum of potassium-deficient female mice

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(6):E1037-E1045
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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