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Neuroendocrinology |
Centro de Producción y Experimentación Animal,3 Universidad de Sevilla, Sevilla, Spain
Instituto de Investigaciones Químicas,4 Centro de Investigaciones Científicas Isla de la Cartuja, 41092 Sevilla, Spain
Department of Anaesthetics, University of Melbourne and Department of Obstetrics and Gynaecology,5 Royal Women's Hospital, Carlton, Victoria 3053, Australia
Area de Farmacología,6 Departamento de Medicina, Universidad de Oviedo, Oviedo, Spain
Animalario Universitario,7 Universidad de Oviedo, Oviedo, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, female reproductive tract, fertilization, neuropeptides, polypeptide receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SP and NKA are encoded by the preprotachykinin-A (PPT-A) gene [8–10]. Four isoforms of mRNA (
, ß, 
, and 
) that result from the alternative splicing of the primary PPT-A transcript have been described. The SP sequence is encoded by all four isoforms, whereas the NKA precursor sequence is present only in ß and PPT-A mRNAs [2, 8–10]. NKB is the only known sequence encoded by the preprotachykinin-B (PPT-B) gene [10, 11]. HK-1 is the product of a third preprotachykinin gene, named PPT-C [6, 7].
Tachykinins interact with three different types of tachykinin receptors, termed NK1, NK2, and NK3, which are preferentially activated by SP, NKA, and NKB, respectively [2–4, 12, 13]. However, the naturally occurring tachykinins are not highly selective and can activate each of the tachykinin receptors [13, 14]. The tachykinin receptors belong to the superfamily of G protein membrane-coupled receptors and have been cloned in different species including the mouse, rat, and human [12, 15–18].
Accumulating data suggest that tachykinins may play a role in the regulation of reproductive functions [4, 11, 19–22]. In the mammalian female reproductive tract, SP and NKA are localized in capsaicin-sensitive primary afferent fibers, the presence of which has been demonstrated in virtually all mammalian species examined [23–25]. Nerve terminals are associated with the myometrial and vascular smooth muscle and are distributed throughout the endocervix [19, 23–25]. Tachykinins have a direct contractile effect in uterine smooth muscle from different species, including humans [20–22, 24–32]. The three tachykinin receptors are expressed in the rat uterus, and their expression and function are selectively and differentially regulated by ovarian steroids [22, 30–32]. Moreover, recent studies have shown that PPT-B, the gene that encodes NKB, is expressed in the uterus [33, 34], which is, together with the placenta [11], one of the few known peripheral tissues that express this gene.
In the present study, we examined the gene expression of tachykinins and tachykinin receptors in different types of reproductive cells from mice. In addition, we performed in vivo studies in mice and rats to investigate the role of tachykinins in the regulation of female reproductive function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experiments were conducted in accordance with NIH guidelines for the care and use of laboratory animals. Wistar rats and Swiss OF-1 mice were purchased from Charles River Laboratories (Criffa, Spain). Animals were maintained in an air-conditioned room at 22°C under controlled lighting (12L:12D) and provided with food and water ad libitum.
Isolation of Cells and Tissues from OF-1 Mice
Female mice (6–9 wk old) received i.p. injections of 100 µl of saline containing 7.5 IU eCG (Intervet, Boxmeer, The Netherlands) followed 46 h later by the same amount of saline containing 7.5 IU of hCG (Intervet). About 17 h later oocyte-cumulus complexes were released from oviducts into M2 medium (Sigma, St. Louis, MO) containing 300 mg/ml hyaluronidase (type IV-S, Sigma), as described by Hogan et al. [35]. After shedding all the cumulus cells, the oocytes were picked up with sterile transfer pipettes and transferred to a Petri dish containing fresh M2 medium. The oocytes were then thoroughly washed until no contaminant cells were present. A similar procedure was used to isolate cumulus cells from superovulated mice. Dispersed cumulus cells were washed several times and placed in M2 medium. Cells were examined under light microscope, and those appearing viable were frozen in liquid N2 until further use. Samples of uterus and brain cortex were rapidly removed from the same mice and dissected free from surrounding tissues. Isolated cells (15–50) or tissue samples (15–10) were rapidly frozen in liquid nitrogen and stored at -80°C until use.
In vivo-fertilized zygotes were isolated from superovulated OF-1 females that were mated with the same strain of males overnight. The following morning, approximately 16 h after hCG injection, the female mice were inspected for vaginal copulatory plugs. Two- and 8- to 16-cell stage embryos were obtained by flushing the oviducts of positive-plugged females 40 and 64 h post hCG, respectively. Blastocysts were obtained flushing both the uterus and oviduct of 90 h post hCG from plug-positive females. The different stages of the embryos were confirmed under light microscope.
Sperm were isolated as described by Hogan et al. [35] for in vitro fertilization. Briefly, male mice were killed and the vas deferentia removed and transferred to a dish containing M2 medium. The sperm were expelled by using a forceps and injection needle. Sperm were maintained during 10 min at 37°C to let the sperm "swim out." After this time period, the supernatant of the dish was collected and centrifuged for 5 min at 735 x g. Sperm were washed twice in M2 medium and frozen.
RNA Extraction and Reverse Transcription-Polymerase Chain Reaction
Reverse transcription-polymerase chain reaction (RT-PCR) reactions were performed essentially as previously described [28]. Total RNA was isolated according to the method of Chomczynski and Sacchi [36]. Residual genomic DNA was removed by incubating the RNA samples with RNase-free, fast protein liquid chromatography pure DNase I (Amersham Biosciences, Essex, UK) and RNasin (Promega Corp., Madison, WI). First-strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase and random hexamers according to the manufacturer's instructions (first-strand cDNA synthesis kit, Amersham Biosciences). The resulting cDNA samples were amplified by PCR using a DNA thermal cycler (MJ Research, Watertown, MA). Amplification of mouse preprotachykinins, tachykinin receptors, neprilysin (NEP), and ß-actin was performed with specific oligonucleotide pairs designed with the analysis software Primer 3 (code available at http://www.genome.wi.mit.edu/genomesoftware/other/primer3) [37]. Amplification of the ß-actin gene transcript was used as an internal control of RT-PCR reactions among the samples. The sequences of the primers and size of the expected PCR fragments are shown in Table 1. All primers were synthesized and purified by Sigma Genosys (Cambridge, UK).
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PCR mixes contained 0.2 µmol primers, 1.5 U of Taq polymerase (Amersham Biosciences), the buffer supplied, 2.5 mmol MgCl2, 200 µmol deoxynucleotide triphosphates, and cDNA in 25 µl. After a hot start (2 min at 94°C), the profile for each cycle was 10 sec at 94°C, 20 sec at 60°C, and 30 sec at 72°C. Cycle numbers were 35 for tachykinins, tachykinin receptors, and NEP and 28 for ß-actin. A higher number of cycles (40) was used in some experiments. Serial half-dilutions of cDNA were amplified at the indicated number of cycles for each target gene and ß-actin to ensure analysis of products in the linear range of amplification. The PCR products were separated by gel electrophoresis on 1.7% agarose, stained with ethidium bromide and visualized and photographed under ultraviolet illumination (Spectronics Corp., New York, NY). The band intensities were scanned by densitometry using a video documentation system and the image analysis software Intelligent Quantifier (BioImage Systems Corp., Ann Arbor, MI). Amplicon sizes were verified by comparison with a DNA mass ladder, and the identity of each PCR product was established by DNA sequence analysis. Minus-RT controls permitted to rule out genomic contamination. Similarly, no products were detected when the RT-PCR steps were carried out with no added RNA, indicating that all reagents were free of target sequence contamination.
Some experiments were performed to analyze the expression of the different PPT-A isoforms in mouse oocytes. The sequence of the primer pair used in these experiments was: forward, 5'-TTTTTCTCGTTTCCACTCAACTG-3' and reverse 5'-ACGCCTTCTTTCGTAGTTCTGC-3'. Experimental conditions were identical to those indicated above except that PCR amplification products were separated by 2.5% agarose gel electrophoresis.
Treatment of Animals with Capsaicin
Neonatal Wistar rats or Swiss OF-1 mice were treated s.c. with capsaicin (50 mg/kg body weight, dissolved in a vehicle of 80% saline, 10% Tween 80, and 10% ethanol) or vehicle in a final volume of 0.05 (mice) or 0.1 ml (rats), on Days 1, 2, 3, 4, and 7 of life. This treatment has been shown to produce selective and permanent degeneration of sensory nerves [38–40]. Hypothermic anesthesia was used in the days of treatment by placing the pups in ice until movements and sensory perceptions were drastically reduced.
The effectiveness of capsaicin treatment in C-fiber denervation was followed by the eye-wipe test. A single drop of capsaicin (0.1% in 90% saline, 10% ethanol) was placed on the eye for a 30-sec period. Eye wiping was almost absent in capsaicin-treated animals, compared with a vigorous eye wiping in control animals. The other eye was subject to a single drop of vehicle, which produced no eye-wiping response. The absence of eye-wiping response persisted during all life in animals treated with capsaicin as neonates.
Eight-week-old female mice or 3- or 6-mo-old female rats (capsaicin-treated or vehicle-treated, age-matched controls) were caged with males for a week. Breeding groups typically consisted of three females and one male. When appropriate, vaginal smears were taken and examined microscopically to assess the stage of the estrous cycle or the presence of copulatory plugs. Only animals with two consecutive regular cycles were used in these experiments.
Treatment of Adult Animals with Tachykinin Antagonists
Eight-week-old female Swiss OF-1 mice were treated with SR 140333 (selective antagonist of the tachykinin NK1 receptor) [41], SR 48968 (selective antagonist of the tachykinin NK2 receptor) [42], or SR 142801 (selective antagonist of the tachykinin NK3 receptor) [43] or solvent (adjusted to a final volume of 10 ml/kg). Each antagonist was injected i.p. at an initial dose of 6 mg/kg for the first day followed by a dose of 3 mg kg day during the next 4 days. The doses administered were those most usually reported in the bibliography [41–44]. From the second to the fifth day, and 2.5 h after injection, females were caged with untreated males for a 4-h period. Females were then checked for a vaginal plug.
In another set of experiments, 6-mo-old female Wistar rats were treated with the selective tachykinin NK2 receptor antagonist SR 48968 (6 mg kg day, 1 ml/kg), the selective tachykinin NK3 receptor antagonist SR 142801 (6 mg kg day, 1 ml/kg) or vehicle (1 ml/kg). The antagonist was injected s.c. in the back during 5 days. Female rats were then mated with untreated males as described above for mice.
Drugs
Capsaicin was purchased from Sigma. SR 140333 (Nolpitantium, (S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl)-4-phenyl-1-azoniabicyclo [2.2.2]octane chloride), SR 48968 (Saredutant, (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide) and SR 142801 (Osanetant, (R)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide) were kindly donated by Dr. X. Emonds-Alt (Sanofi-Synthelabo, Montpellier, France).
Statistical Analysis
Data are expressed as means ± SEM, and n represents number of experiments. Each animal was used only once. Statistical analysis of litter sizes was performed by Student t-test. Number of females delivering litters (reproductive success) was compared using the chi-square test. A probability level of P< 0.05 was regarded as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1 shows the gene expression of tachykinins, tachykinin receptors, and neprilysin in different types of reproductive cells from mice. The preprotachykinin genes PPT-A, PPT-B, and PPT-C that encode SP/NKA, NKB, and HK-1, respectively, and the genes that encode the tachykinin NK1, NK2 and NK3 receptors were all expressed, at different levels, in the uterus of superovulated, unfertilized mice. The mRNA of NEP, the main enzyme involved in tachykinin degradation at the uterine level, was also expressed in the uterus. The PCR product observed for each target gene had the predicted size (Table 1 and Fig. 1), and its identity was confirmed by DNA sequence analysis. The tachykinin precursors, the three tachykinin receptors, and NEP were also found in the mouse cerebral cortex, used as a positive control of amplification of the target genes (Fig. 1).
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Isolated cumulus cells expressed PPT-A, PPT-B, PPT-C, and NEP and low levels of tachykinin NK1 and NK2 receptors. Mouse oocytes expressed significant amounts of PPT-A and PPT-B mRNA transcripts. By using the appropriate primer pair and 2.5% agarose gel electrophoresis, we observed that, among the four PPT-A isoforms, the ß-isoform was the most abundantly expressed, with a lower expression of the mRNA isoform (not shown). A low but clearly detectable expression of the three tachykinin receptors was observed, but HK-1 and NEP were undetectable even after 40 cycles of PCR (Fig. 1).
We failed to detect the presence of most of the mRNAs of the different target genes in 2- and 8–16-cell mouse embryos, which expressed only a very low-abundance transcript corresponding to the NK1 receptor, even after 40 cycles of PCR amplification. However, the mRNAs of NKB, HK-1, and NEP mRNA transcripts appeared in blastocyst-stage embryos. A single transcript of low abundance, corresponding to the NK2 receptor, was the only target gene detected in mice sperm (Fig. 1).
Effect of Capsaicin in Fertility
Female mice treated with capsaicin as neonates showed an apparently normal courtship behavior after caging with males during a week. However, the reproductive success was lower in capsaicin-treated animals, compared with control, vehicle-treated animals (Fig. 2A). The litter size was not significantly reduced in females treated with capsaicin (Fig. 2B).
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In our hands, treatment of newborn rats with capsaicin affected neither body weight nor estrous cyclicity, which were similar in capsaicin- and vehicle-treated animals (data not shown). Female rats treated neonatally with capsaicin showed an apparently normal courtship behavior but exhibited a lower reproductive success and litter size, compared with control animals (Fig. 3). At 3 mo of age, 4 of 18 rats became sperm positive during a 1-wk exposure to males, and only one of these sperm-positive rats delivered a litter (Fig. 3A). At 6 mo of age, 4 of 12 female rats became pregnant (Fig. 3B) and delivered litters of reduced size (Fig. 3C).
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Effect of Tachykinin Receptor Antagonists in Fertility
Treatment of 8-wk-old mice with the selective tachykinin NK1 receptor antagonist SR 140333, the selective tachykinin NK2 receptor antagonist SR 48968, or the selective tachykinin NK3 receptor antagonist SR 142801 (each antagonist at a dose of 6 mg/kg for the first day, 3 mg kg day during the following 4 days) had no effect on reproductive success or litter size (Fig. 4).
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In 6-mo-old female rats, treatment with the tachykinin NK2 antagonist SR 48968 had no effect on fertility (Fig. 5). Treatment with the tachykinin NK3 antagonist SR 142801 did not significantly decrease the reproductive success but reduced the litter size of female rats (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The tachykinins have been described almost exclusively as peptides of neuronal origin. SP and NKA are present in the central nervous system and also in sensory nerves innervating peripheral tissues [1–5, 13, 19]. These tachykinins are released from nerve endings at both the spinal cord and the peripheral level, playing a role as excitatory neurotransmitters [1–5]. NKB has been detected almost exclusively at the level of the central nervous system [10, 11, 13, 40, 45]. However, it has been shown that SP is also expressed in nonneuronal cells, such as human and mouse Leydig cells [46] and different types of human immune cells [47]. Recent reports have also indicated the presence of the NKB mRNA in the human and rat placenta [11] and in the rat uterus [33, 34]. Moreover, a recently cloned new member of the tachykinin family, HK-1 [6], was primarily detected in nonneuronal cells [6, 7]. These data suggest that tachykinins have a widespread distribution, not merely restricted to neuronal tissues. In the female reproductive tract, immunohistochemical studies demonstrated that SP and NKA are stored in capsaicin-sensitive, primary afferent neurons supplying the uteri of several species including the mouse, rat, and human [19, 23–25]. The present results show that the PPT-A mRNA was expressed in the mouse uterus, cumulus cells, and unfertilized oocytes. The PPT-B mRNA was detected in the uterus, cumulus cells, unfertilized oocytes, and blastocyst-stage embryos. The PPT-C mRNA was found in the uterus, granulosa cells, and blastocyst-stage embryos. The three tachykinin receptors and NEP, the main enzyme involved in tachykinin metabolism, were also expressed, at different levels, throughout the female reproductive tract [20–22, 27–31, this study]. These data show that in addition to its neuronal location, tachykinins are present in different types of reproductive cells of nonneuronal origin and may play a role as intercellular signaling molecules in female reproductive function.
The present study demonstrates that mouse unfertilized oocytes expressed the PPT-A mRNA transcript. Among the four PPT-A isoforms, the ß mRNA isoform was the most abundant, with a lower expression of the isoform. These are the two PPT-A isoforms that encode both SP and NKA, suggesting that the two tachykinins may be present in oocytes. Unfertilized oocytes also expressed PPT-B and low levels of the three tachykinin receptors. With the exception of a low-abundance transcript, corresponding to the tachykinin NK1 receptor, none of the target genes were detected from the 2-cell to the 8- to 16-cell stage embryos. Taken into account that, in the mouse, the genome of the embryo becomes transcriptionally competent at the end of the one-cell stage [48], our data show that: 1) with the exception of the tachykinin NK1R, the tachykinin and tachykinin receptor transcripts are of maternal origin and not transcribed at the onset of zygotic transcription and 2) tachykinins are probably not involved in the morphogenetic transitions that occur during preimplantation. The appearance of PPT-B, PPT-C, and NEP mRNA transcripts in blastocyst stage embryos argue for a possible role of these tachykinins in the implantation process, when the embryo comes into direct contact with the uterus. With respect to NKB, these data support previous findings by Page et al. [11], which showed the presence of NKB in the human and rat placenta and suggested that this tachykinin could play a role in trophoblast invasion occurring during implantation.
It is axiomatic that functional correlates of receptor activation may be inferred from tissue distribution of that receptor. In vivo studies were then performed in mice and rats to analyze more directly the role of neuronal and nonneuronal tachykinins in reproductive function. Chronic administration of capsaicin to a variety of animal species results in a functional impairment of afferent nerve activity, principally mediated via its action on vanilloid VR1 receptors on these nerves [38, 39, 49, 50]. Treatment of mouse or rat neonates with capsaicin results in an appreciable decrease in fertility, suggesting that capsaicin-sensitive sensory nerves could play a role in regulating fertility in females [19, 50, this study]. With regard to our results in mice and rats, the block of tachykinin receptors could not explain, at least completely, the effects of capsaicin on fertility. A possible explanation of these data is that, in addition to tachykinins, other neuropeptides present in capsaicin-sensitive sensory neurons, such as calcitonin-gene related peptide, cholecystokinin, nitric oxide, or others [24, 25, 51] could be involved in the effects of capsaicin. An alternative hypothesis, derived from the enormous redundancy of peptide transmitters and their receptors in one animal species, is that the block of a system could be amended by activation of neighboring, backup systems. In addition, it should be noted that the effects of SR 140333, SR 48968, and SR 142801 are species dependent and none of these tachykinin antagonists are able to cross the blood-brain barrier [41–44]. Therefore, they cannot mimic the possible central component of the effects of capsaicin in fertility.
The present study shows that rats treated with SR 142801 exhibited a tendency toward a decreased fertility, with a reduction of litter size. It must be noted that: 1) although absent in most peripheral tissues, the tachykinin NK3 receptor is expressed in female reproductive tissues from different species [4, 22, 26, 32, 52]; 2) NKB, previously thought to be present exclusively in the central nervous system, is expressed in the placenta, uterus, and oocytes [11, 33, 34, this study]; 3) at least in the rat uterus, the expression of both NKB and the tachykinin NK3 receptor is under estrogen control [31–34]; 4) the levels of expression of NKB and the NK3 receptor in the female reproductive system are maximal around implantation [11, 22]; and 5) the only identified sexually dimorphic population of neurons in ovine females express NKB [53]. Collectively, these data argue for an important and, probably, sexually dimorphic role of the NKB/tachykinin NK3 receptor ligand receptor pair in female reproduction.
Finally, a low-abundance band corresponding to the NK2R mRNA was the only transcript expressed in mouse sperm. These data support recent findings showing that spermatozoa contain their own repertoire of mRNAs [54]. SP is expressed in Leydig cells of human and mouse testes [46], and it has recently been shown that tachykinin NK1R and NK2R are expressed in the human corpus cavernosum [55]. It would therefore be of interest to investigate further the role of tachykinins in male reproductive function.
In conclusion, the results of early immunohistochemical studies [23–25] and the present study show that tachykinins of both neuronal and nonneuronal origin are expressed in different types of cells involved in reproduction. These findings and those in animals treated in vivo with capsaicin or SR 142801 suggest that these peptides may be involved in the regulation of female reproductive function. Further studies are needed to determine the precise role of tachykinins in oocytes and embryo development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: M.L. Candenas, Instituto de Investigaciones Químicas, Avenida Americo Vespucio, s/n, Isla de la Cartuja, 41092 Sevilla, Spain. Fax: 34954460565; mluz{at}cica.es
Received: 13 March 2003.
First decision: 8 April 2003.
Accepted: 30 April 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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