Neuropeptide effect on splenocytes modifies ovarian steroidogenesis

SECRETION FROM NEUROPEPTIDE-TREATED SPLENOCYTES MODIFIES OVARIAN STEROIDOGENESIS

LILIANA OLIVEROS, MYRIAM FORNERIS, LUIS AGUADO*

Laboratorio de Biología de la Reproducción, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, San Luis

Resumen

Secreciones de esplenocitos tratados con neuropéptidos modifican la esteroidogénesis ovárica.  Algunas evidencias indican que la función inmune está regulada por el sistema nervioso simpático y su neurotransmisor principal, norepinefrina (NE), a través del sistema nervio ovárico superior (NOS)-ganglio celíaco-inervación posganglionar noradrenérgica del bazo. En el presente trabajo, se seccionó el NOS a ratas de 53 días de edad y se sacrificaron los animales siete días más tarde (60 días de edad). Se aislaron los esplenocitos del bazo y se cultivaron por 48 hs. Se trabajó paralelamente con ratas controles (con operación simulada). El medio de cultivo se usó para estimular ovarios de ratas intactas de 60 días en diestro 2 (sin el NOS seccionado o con operación simulada) en incubaciones in vitro. Se observó disminución de la progesterona y aumento del estradiol liberados por el ovario, en relación al efecto producido por el medio de cultivo de esplenocitos de ratas controles. Cuando los esplenocitos de ratas con el NOS seccionado (NOS-s) fueron tratados con péptido intestinal vasoactivo (VIP) o con neuropéptido Y (NPY), 10-6M durante 24 h, sus secreciones incrementaron la liberación de progesterona, mientras que disminuyeron la de estradiol, desde ovarios intactos, comparado con las secreciones de los esplenocitos no tratados de ratas NOS-s. Las secreciones de esplenocitos tratados con VIP disminuyeron la liberación de androstenodiona desde ovarios intactos, mientras que con NPY no se observaron cambios significativos. La respuesta esteroidogénica ovárica, que fue modificada por los efectos de la sección del NOS sobre las células del bazo, fue revertida cuando los esplenocitos de ratas NOS-s fueron tratados in vitro con VIP or NPY. Esto sugiere que la sección del NOS disminuye la llegada de VIP y NPY al bazo. Cuando se adicionan dichos neuropéptidos a los esplenocitos en cultivo, las secreciones de estas células revierten  el perfil de las hormonas liberadas por ovarios intactos. En este trabajo también se muestra evidencia de la modulación de la función inmune por el sistema nervioso simpático y por  neurotransmisores distintos a NE.

Palabras clave:  esplenocitos, péptido intestinal, vasoactivo, neuropéptido Y, esteroidogénesis ovárica

Abstract

There are evidences for modulation of immune function by the sympathetic nervous system and its principal neurotransmitter norepinephrine (NE) through superior ovarian nerve (SON)-coeliac ganglion-noradrenergic postganglionic innervation of the spleen. Seven days after SON transection at 53 days of age, the rat splenocytes were isolated and then cultured for 48 h. These culture media, used to stimulate ovaries from 60-day-old intact rats (neither SON-transected nor sham-operated) at diestrus 2 stage, in in vitro incubations, showed a decrease in progesterone release, an increase in estradiol release and no change in androstenedione release in relation to splenocyte culture media from control (sham-operated) rats. When esplenocytes from SON transected (SON-t) rats were treated with vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY), both at 10-6M for 24 h, their secretions increased the progesterone release while decreasing the estradiol release from the intact ovaries, compared with the secretions of untreated splenocytes from SON-t rats. Although the secretions of splenocytes treated with VIP decrease the androstenedione release from the ovaries, the treatment with NPY produced no change in hormone release. In the present paper the ovarian steroidogenic response, which was modified by the effects of an in vivo SON transection on spleen cells, was reverted by an in vitro system in which the splenocytes were treated with VIP or NPY. This could indicate that the spleen of SON-t rats does not receive those neuropeptides by neural route however, when they are added to splenocyte culture in vitro, the cell secretions revert the profile of steroid hormones released from the intact ovary. We also present functional evidence for modulation of the immune function by sympathetic nervous system and neurotransmitters other than NE.

Key words: splenocytes, vasoactive intestinal peptide, neuropeptide Y, ovarian steroidogenesis.

Communication between the neuroimmune and endocrine systems and a reciprocal flow of information are suggested by several observations1. It is known that sympathetic noradrenergic (NA) nerve fibers and sensory neurons inervate immune organs such as the spleen, the thymus, the lymph nodes and gut-associated lymphoid tissues2-4. On the other hand, the ability of various hormones to affect immune functions in vivo and in vitro has long been known. An elevation of adrenal glucocorticoids during stress has been considered to be responsible for stress-induced immunosuppression5. LH, FSH, PRL, GH, TSH, among others, have been shown to modulate the immune response6, 7.

The noradrenergic postganglionic innervation of rat spleen originates mainly in the superior mesenteric/coeliac ganglion and the nerve fibers enter the spleen at the hilar region with the vasculature8, 9. Electron microscopic immunocytochemical studies of tyrosine hydroxylase nerve fibers have shown direct contacts of nerve terminals with lymphocytes and macrophages in the spleen10. The endings of these nerves contain norepinephrine (NE), and, among other neuropeptides, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY)11, 12. The presence of  NE, VIP and NPY receptors in rat splenic lymphocytes has been demonstrated12-14. The reduction in splenic NA innervation is associated with degeneration of NA nerve fibers and ultimately, with loss of their cell bodies in the superior mesenteric/coeliac ganglionic complex15.

On the other hand, it is known that the superior ovarian nerve (SON), which innervates the ovary, has its preganglionic cell bodies in the spinal cord16. These exit at thoraco spinal cord at levels T10 and T11 and synapse in neurons of coeliac and suprarrenal ganglia. It has been demonstrated that the SON contains mainly afferent and efferent noradrenergic (NA) fibers and also vipiergic fibers from the coeliac ganglion17-19. SON transection in adult rats affects splenocyte secretions, which participate in the ovarian steroidogenic response, particularly in progesterone release, which secretions might be controlled by adrenergic influences since the number of splenocyte b-adrenergic receptors changes through SON-coeliac ganglion-noradrenergic postganglionic innervation of the spleen20. There is almost no  information about the effect of SON transection on the spleen cells, which, like the ovary, are also innervated extensively by afferent and efferent noradrenergic sympathetic nerve fibers from the coeliac ganglion16, 17.

The aim of the present work was to study if the effects of an in vivo SON transection on the spleen cells, determined by the effect of the secretions of those cells on the steroidogenic response of intact ovaries, could be reverted by an in vitro system in which the splenocytes were treated with VIP or NPY, as we have previously done with NE20. Culture media of splenocytes from SON transected (SON-t) rats and of those splenocytes treated with VIP or NPY were used to stimulate intact ovaries.

Materials and Methods

Chemicals. Liquid culture medium, penicillin G, streptomycin, crude trypsin, soybean trypsin inhibitor, VIP and NPY were purchased from Sigma Chemical Co (St. Louis, MO, USA).  Progesterone, estradiol, androstenedione were provided by New England Nuclear (Boston, MA, USA). All the other chemicals were of reagent grade and were purchased from Merck Laboratory, Buenos Aires, Argentina.

Animals and experimental model. Adult female Holtzman rats were used. They were housed in a controlled environment (22-24 oC, 12 h light- 12 h dark). Water and food were available ad libitum. Two groups of cyclic adult rats were used, one SON-transected (SON-t) and the other one sham-operated (control) at 53 days of age. Seven days after the SON transection was performed all rats were killed and  the splenocytes were isolated. The splenocytes from SON-t and control rats were preincubated for 24 h, then, the medium was removed and the cells were cultured for an additional 48 h. On the other hand, splenocytes from SON-t rats were cultured in presence of VIP in one case and NPY in another, both at 10-6 M, for 24 h. After that, the medium was replaced by fresh medium and the splenocytes were cultured for an additional 24 h. Fluids were collected and used to stimulate ovaries from 60-day-old intact rats (neither SON-t nor sham-operated) rats in diestrus 2 stage, in in vitro incubation to measure the steroidogenic response (Fig.1). Considering the influence of SON transection on both the ovary and the spleen, via the coeliac ganglion, in all the experiments carried out, the effect of the culture medium from splenocytes was studied on ovaries from intact rats.

Transection of the superior ovarian nerve. Rats were lightly anaesthetized with ether and the ovaries were exposed through bilateral dorsal incisions. The SON transection was achieved as described previously20. In brief, the suspensory ligament enclosing the SON was lifted with fine forceps, the nerve was cut with  small scissors, the ovaries were returned to the abdominal cavity, and the wounds were sutured. The suspensory ligament is clearly visible and the SON travels along it, thus, we assumed that transection of the suspensory ligament results in transection of the SON.

Splenocyte cultures. In brief, rats were killed by decapitation, their spleens were aseptically removed, washed in saline solution and pressed through a sterile nylon screen (200 mm mesh) to obtain individual splenocytes. The cells were collected by centrifugation (600 g, 5 min,) resuspended in serum-free RPMI medium and treated with 0.83% NH4Cl: 2.6% TRIS (9:1, v/v) pH 7.2, to remove the red blood cells. Cells (1 x 106 viable cells/ml of medium) were cultured in 8 x 75 mm  glass test tubes at 37 oC in 95% air: 5% CO2 atmosphere. The medium was supplemented with 10% fetal bovine serum and  antibiotics (50 mg/ml streptomycin and 50 units/ml penicillin).

Ovary  incubation. After killing the intact rats, the ovaries were immediately removed, dissected, weighed and preincubated in 1 ml of fresh culture medium at 37 oC, in 95% O2: 5% CO2 mixture. After 30 min, the medium was discarded and 1 ml of splenocyte culture medium was added. Incubation was continued for 3 h and then the medium was removed and stored at -70 oC for subsequent measurement of steroids.

Steroid assays. The progesterone, estradiol and andros-tenedione contents of culture media were determined by specific RIAs using antisera as described previously21. The assay sensitivity  was less than 5 ng/ml for progesterone, 12 fmole/tube for estradiol and 0.02 ng/ml for androstenedione. The  inter- and intraassay coefficients of variation for all the assays were less than 10.0%.

Statistical analyses. Results are expressed as mean ± SEM. Significant differences among means were considered at a level of P< 0.05 and tested by Student’s t-test.

Results

The effect of secretions from splenocytes treated with neuropeptides on the ovarian steroidogenesis is shown in the figures. The results were expressed in all cases as ng or pg / mg ovary or / mg protein. Fig. 2 shows the progesterone release from ovaries of control and SON-t rats incubated with culture medium of splenocytes. When culture medium of splenocytes from SON-t rats was used, the progesterone release from ovaries decreased significantly in relation to that observed when the splenocyte culture medium came from control rats, p< 0.001. When splenocytes from SON-t rats were treated with VIP or NPY, their secretions increased the progesterone release from the ovaries (p< 0.001), compared with the secretions from SON-t rats untreated splenocytes. The values obtained in presence of neuropeptides reached the progesterone levels released by the ovaries incubated with culture medium from splenocytes of control rats (p< 0.001).

As shown in Fig. 3, the estradiol release from ovaries incubated with culture medium of SON-t rat splenocytes without neuropeptide treatment increased markedly in relation to that obtained with culture medium of splenocytes from control rats, p< 0.001. However, when  culture medium of SON-t rat splenocytes treated with VIP or NPY was used, a significant  decrease (p< 0.001) in estradiol release from ovaries was observed, compared with that from ovaries incubated with secretions of SON-t rat untreated splenocytes.

When culture medium of splenocytes from SON-t rats was used, the androstenedione release from ovaries did not change in relation to that observed when the splenocyte culture medium derived from control rats. Similar results were obtained  when splenocytes from SON-t rats were previously treated with VIP. The secretions of splenocytes treated with NPY decreased (p< 0.001) the androstenedione release from the ovaries (Fig. 4).

Discussion

The ovary and spleen are innervated extensively by afferent and efferent noradrenergic  sympathetic nerve fibers from the coeliac ganglion16, 17, 22, 23. The endings of those nerves contain NE, VIP and NPY11, 12, among other neurotransmitters. In particular, the coeliac ganglion is connected through the superior ovarian nerve to the ovary 16, 17, 24.  We have previously shown that the effects on the splenocyte b-adrenergic receptors number after an in vivo SON transection were simulated by an in vitro system modulating b-adrenergic receptors number by treatment of splenocytes from SON-t rats with and without NE. Additionaly, we have observed that the change in the ß-adrenergic receptors number was associated to a change in the secretion of those splenocytes since ovaries from intact rats incubated with those secretions modify their steroidogenic response. It was suggested that the activity of splenocytes could be controlled by adrenergic influences20. In the present investigation we showed that the effects of an in vivo SON transection on the splenocyte secretions could be reverted by an in vitro system in which the splenocytes were treated with VIP or NPY, as we have previously done with NE20. We showed that the secretions of the cultured splenocytes from SON-t rats produced a decrease of progesterone, an  increase of estradiol and no change in androste-nedione release from  intact ovaries. It has been observed that circulating levels of IL-1 are elevated during luteal phase of normally cycling women25. Inversely, low concentrations of progesterone appear to upregulate macrofage IL-1 gene expression, while higher concentrations of progesterone significantly  inhibit IL-1 activity26. In our case, low concentrations of progesterone were released from the ovaries when they were incubated with secretions of the cultured splenocytes from SON-t rats, suggesting that those secretions are not the same as those of splenocytes from control rats. This fact could be in agreement with the eventual participation of IL-1 in the process. In an in vivo situation, there is no possibility that high levels of progesterone affect the splenocyte secretions, since the circulating progesterone levels did not change with the SON transection (data not shown). We also show that splenocyte secretions of SON-t rats stimulate estrogen release from the ovaries, indicating that the SON transection modifies the splenocyte secretions. It has been demonstrated in granulosa cells that the steroidogenic activity of a factor obtained from the splenocytes of normal animals could not be attributed to a FSH-like molecule. Those authors suggest that that factor is different from FSH because it does not contain FSH immunoreactivity nor does it contain FSH receptor-binding activity27.

It is known that rat splenic lymphocytes possess VIP and NPY receptors12-14. In this paper we show evidence that splenocyte secretions, which participate in the ovarian steroidogenic response, might be controlled by VIP and NPY. We observed that culture medium of SON-t rats, containing the secretions of VIP treated or NPY treated splenocytes increased the progesterone release from the ovaries, while the release of estradiol decreased, compared with the secretions of untreated splenocytes. On the other hand, the secretions of NPY  treated splenocytes decreased the androstenedione release from the ovaries, but no change was observed when splenocytes were treated with VIP. Probably, the decrease in estradiol release, when ovaries were stimulated with secretions of splenocytes treated with NPY, could be associated with the decrease in androstenedione release in the same conditions, considering that androstenedione is a natural precursor of estradiol. Recently, it has been shown that NPY greatly enhanced IL-4 production and inhibited that of interferon-gamma (IFN-gamma) from mouse helper T cell subsets. However, VIP had no effects on IFN-gamma production, while it inhibited IL-4 production slightly but consistently28. It has been shown that the addition of nerve growth factor (NGF) to unstimulated purified T cells induces the synthesis of NPY29. Thus, these neuropeptides modify the activity of the immune cells. However, the effect of the immune system on ovarian steroidogenic activity is still not clear.

Considering our previous report20 and the results presented in this study, we can speculate that the SON transection could modify the coeliac ganglion, in a retrograde way, affecting the release of  VIP and NPY through its sympathetic fibers to the spleen. This fact could modify the splenocyte activity, probably by releasing specific secretions which, as we have shown, influence the steroidogenic response of intact ovaries. That steroidogenic response was reverted when ovaries were incubated with culture medium of SON-t rats splenocytes treated with VIP or NPY. The factor(s) secreted by splenocytes might provide a link between intraovarian spleen cells and the regulation of the ovarian function. It is known that lymphocytes release specific cytokines which modulate granulosa cell steroidogenesis, typically in an inhibitory30 and stimulatory fashion31, but we cannot affirm that the ovarian steroidogenic-modulating factor(s) produced by the splenocytes treated with VIP or NPY is (are) a cytokine.

The relevance of the factor(s) secreted by the splenocytes to the ovarian physiology remains a matter of study but, it is possible that that secretion, either in the spleen or in the circulation, could affect the ovarian function through an endocrine mechanism.

On the other hand, knowing that immune signals influence the hypothalamic-pituitary axis32 it is possible that the factor(s) secreted by the splenocytes in the intact animal could be released into the general circulation to act on hypothalamic-pituitary sites and subsequently originate modifications in the ovarian function.

Acknowledgements: This work has been supported by Grants 3711/92 from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina) and 9302 from San Luis University, Argentina.

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Fig. 4.- «In vitro» androstenedione release from ovaries of 60-day-old intact rats stimulated with culture medium of splenocytes from control (open bars) and SON-t (solid bars) rats. Treatment of splenocytes with VIP or NPY as well as the number of determinations and experiments were done as indicated in legend of Fig. 2. A) Androstenedione ng / mg ovary. B) Androstenedione ng / mg protein. Values are expressed as the mean ± SEM. Bars sharing a same superscript letter indicate significant differences at a p<0.001 as determined by Student’s t- test.

                                              + VIP

             Culture               from

         splenocytes             SON-t

                          rats             + NPY

     from           from

   SON-t       control

      rats             rats

Incubation of ovaries         Progesterone

from intact rats             Estradiol

                        Androstenedione

Fig. 1.– Diagram of the experimental design.

Fig. 2.– «In vitro» progesterone release from ovaries of 60-day-old intact rats stimulated with culture medium of splenocytes from control (open bars) and SON-t (solid bars) rats. Secretions from SON-t rat splenocytes incubated 24 h with and without VIP or NPY, both at 10 -6M, and then cultured in fresh medium alone for another 24 h were used. A) Progesterone ng / mg ovary. B) Progesterone ng / mg protein. Values are expressed as the mean ± SEM of triplicate determinations of 2 different experiments, using 4 rats per group per experiment (total 16 ovaries per group). Bars sharing a same superscript letter indicate significant differences at a,b,c p<0.001 as determined by Student’s t- test.

Fig. 3.- «In vitro» estradiol release from ovaries of 60-day-old intact rats stimulated with culture medium of splenocytes from control (open bars) and SON-t (solid bars) rats. Treatment of splenocytes with VIP or NPY as well as the number of determinations and experiments were done as indicated in legend of Fig. 2. A) Estradiol pg / mg ovary. B)  Estradiol pg / mg protein. Values are expressed as the mean ± SEM. Bars sharing a same superscript letter indicate significant differences at a,b,c p<0.001 as determined by Student’s t- test.

Received: 4-I-2000

Accepted: 2-XI-2000

*Member of Research Career of CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas)

Postal address: Dr. Luis Aguado, Laboratorio de Biología de la Reproducción, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina.

Fax: 54-2652-430224                e-mail: labir@unsl.edu.ar