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Kisspeptin, the product of the KISS1 gene, plays an essential role in the regulation of spermatogenesis acting primarily at the hypothalamic level of the gonadotropic axis. However, the presence of kisspeptin and its canonical receptor, KISS1R, in spermatozoa has not been explored nor the direct effects of kisspeptin on sperm function have been studied so far. In the present study, we analysed the expression of kisspeptin and its receptor in sperm cells by western blot and immunocytochemistry assays and evaluated the effects of exposure to kisspeptin on sperm intracellular Ca(2+) concentration, [Ca(2+)]i, sperm motility, sperm hyperactivation and the acrosome reaction. Changes in [Ca(2+)]i were monitored using Fura-2, sperm kinematic parameters were measured using computer-assisted sperm analysis (CASA), and the acrosome reaction was measured using fluorescein isothiocyanate-coupled Pisum sativum agglutinin lectin (FITC-PSA method). We found that kisspeptin and its receptor are present in sperm cells, where both are mainly localized in the sperm head, around the neck and in the flagellum midpiece. Exposure to kisspeptin caused a slow, progressive increase in [Ca(2+)]i, which reached a plateau about 3-6 min after kisspeptin exposure. In addition, kisspeptin modulated sperm progressive motility causing a biphasic (stimulatory and inhibitory) response and also induced transient sperm hyperactivation. The effects of kisspeptin on sperm motility and hyperactivation were inhibited by the antagonist of KISS1R, peptide 234. Kisspeptin did not induce the acrosome reaction in human spermatozoa. These data show for the first time that kisspeptin and its receptor are present in human spermatozoa and modulate key parameters of sperm function. This may represent an additional mechanism for their crucial function in the control of male fertility.

Studies were undertaken to determine the nature of the receptors mediating contractile effects of tachykinins in the uteri of nonpregnant women, and to analyse the expression of preprotachykinins (PPT), tachykinin receptors and the cell-surface peptidase, neprilysin (NEP), in the myometrium from pregnant and nonpregnant women. The neurokinin B (NKB) precursor PPT-B was expressed in higher levels in the myometrium from nonpregnant than from pregnant women. Faint expression of PPT-A mRNA was detectable in the myometrium from nonpregnant but not pregnant women. PPT-C, the gene encoding the novel tachykinin peptide hemokinin-1 (HK-1), was present in trace amounts in the uteri from both pregnant and nonpregnant women. Tachykinin NK2 receptors were more strongly expressed in tissues from nonpregnant than from pregnant women. NK1 receptor mRNA was present in low levels in tissues from both pregnant and nonpregnant women. A low abundance transcript corresponding to the NK3 receptor was present only in tissues from nonpregnant women. The mRNA expression of the tachykinin-degrading enzyme NEP was lower in tissues from nonpregnant than from pregnant women. Substance P (SP), neurokinin A (NKA) and NKB, in the presence of the peptidase inhibitors thiorphan, captopril and bestatin, produced contractions of myometrium from nonpregnant women. The order of potency was NKA≫SP≥NKB. The potency of NKA was unchanged in the absence of peptidase inhibitors. The tachykinin NK2 receptor-selective agonist [Lys5MeLeu9Nle10]NKA(4–l0) was approximately equipotent with NKA, but the tachykinin NK1 and NK3 receptor-selective agonists [Sar9Met(O2)11]SP and [MePhe7]NKB were ineffective in the myometrium from nonpregnant women. The uterotonic effects of [Lys5MeLeu9Nle10]NKA(4–10) were antagonized by the tachykinin NK2 receptor-selective antagonist SR48968. Neither atropine, nor phentolamine nor tetrodotoxin affected responses to [Lys5MeLeu9Nle10]NKA(4–10). These data are consistent with a role of tachykinins in the regulation of human uterine function, and reinforce the importance of NK2 receptors in the regulation of myometrial contraction. Keywords: Human myometrium, [Lys5MeLeu9Nle10]NKA(4–10), NK1 and NK2 receptors, neprilysin, neurokinin A, neurokinin B, substance P, tachykinins, uterine contractions, SR48968 Introduction Tachykinins are members of a family of neuropeptides that includes substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). Their biological actions are mediated through three receptors belonging to the family of G protein-coupled receptors (GPCR), denoted NK1, NK2 and NK3, that have highest affinity for SP, NKA and NKB, respectively (Henry, 1986; Regoli et al., 1994a,1994b; Lecci et al., 2000). SP and NKA, but not NKB, are present within capsaicin-sensitive sensory nerves in the periphery and are locally released by a number of physical and chemical stimuli (Holzer, 1988; Maggi & Meli, 1988). SP and a new member of the tachykinin family, hemokinin-1 (HK-1), that has recently been cloned in the mouse (Zhang et al., 2000), rat and human (Kurtz et al., 2002) are also produced in non-neuronal cells (Joos & Pauwels, 2000; Zhang et al., 2000). This distribution suggests an important role for tachykinins in intercellular communication. Previous studies from other laboratories and from ours indicate a role of tachykinins in the regulation of uterine function. Early immunohistochemical studies showed the presence of tachykinin-immunoreactive nerves supplying the uteri of several species including the mouse, rat, guinea-pig and human (Alm et al., 1978; Huang et al., 1984; Traurig et al., 1984; Papka et al., 1985; Samuelson et al., 1985; Heinrich et al., 1986; Alm & Lundberg, 1988; Reinecke et al., 1989; Traurig et al., 1991; Papka & Shew, 1994). The association of these nerves with smooth muscle indicates that tachykinins released from their peripheral terminals may influence uterine contractility. Preprotachykinin-A (PPT-A), the precursor of SP and NKA, is also expressed in non-neuronal cells such as monocytes and macrophages (Ho et al., 1997); these can be associated with the mammalian uterus (Cocchiara et al., 1997). SP has also recently been reported to participate in stress-induced abortion in the mouse and possibly in the human (Arck et al., 1995; Markert et al., 1997; Marx et al., 1999; Joachim et al., 2001). NKB has recently been reported to be expressed in the human placenta (Page et al., 2000) and the gene that encodes it, preprotachykinin-B, is expressed in the uterus of the rat and the human (Pinto et al., 2001;2002). NKB has been implicated in the symptoms associated with pre-eclampsia (Page et al., 2000). The major tachykinin-degrading enzyme neprilysin (NEP) (Matsas et al., 1983;1984; Hooper et al., 1985; Hooper & Turner, 1985) is also present in the uterus (Ottlecz et al., 1991; Head et al., 1993; Riley et al., 1995). This intrauterine distribution supports the view that tachykinins may be important intercellular signalling molecules within the female reproductive tract (Pinto et al., 2001; 2002). To date, the majority of molecular (Pinto et al., 1999; Candenas et al., 2001), immunohistochemical (Traurig et al., 1984;1991; Papka et al., 1985) and functional (Fisher et al., 1993; Pennefather et al., 1993b; Fisher & Pennefather, 1997; Magraner et al., 1998; Patak et al., 2000a) studies of the uterine distribution of tachykinins, tachykinin receptors and/or degradation of tachykinins have been conducted using the rat. It is now emerging, however, that there are species (Patak et al., 2000a,2000b;2002a) as well as hormonal- and pregnancy-related differences in tachykinin expression and actions in the mammalian myometrium (Pinto et al., 1999;2001;2002; Candenas et al., 2001). We have previously described the uterotonic effects of tachykinins on myometrium of late-pregnant women (Patak et al., 2000b). However, no systematic molecular studies of the expression of tachykinin precursor genes, neither of tachykinin receptors nor of NEP in either pregnant or nonpregnant human uterus, have been undertaken to date. Moreover, despite early reports that SP and eledoisin elicit contractions of the nonpregnant human uterus (Molina & Zappia, 1976; Ottesen et al., 1983), there have been no systematic functional studies of the actions of tachykinin peptides on myometrium from nonpregnant women. For these reasons, the aim of the present study was to examine further the tachykinins, their receptors and NEP on the human myometrium. We have therefore performed molecular studies using myometrium from both nonpregnant and pregnant women, and functional studies to characterize the effects of tachykinins on uterine contraction of tissue from nonpregnant women. A preliminary account of some of the results of this study has been presented previously (Patak et al., 2002b; Pinto et al., 2002).

The complete A + T-rich region of mitochondrial DNA (mtDNA) has been cloned and sequenced in the species of the Drosophila subobscura subgroup D. subobscura, D. madeirensis and D. guanche. Comparative analysis of these sequences with others already published has identified new sequence motifs that are conserved in Drosophila and other insects. A putative bi-directional promoter and a stop signal are proposed to be involved in the primary mtDNA strand replication of Drosophila. This region strongly resolves relationships of the species included in a phylogenetic analysis, both for closely related species and also at deeper phylogenetic levels when only the left and central domains are taken into account.

Although the Canary Islands were settled by humans, possibly of Berber origin, as late as 2500 years ago, the precise course and numbers of early migrations to the archipelago remain controversial. We have therefore analysed mtDNA variation (HVS-I as well as selected RFLP sites) in 300 individuals from the seven Canary Islands. The distribution and variation across the islands in a specific mtDNA clade of Northwest African ancestry suggest that there was one dominant initial settlement process that affected all the islands, from east to west. This indicates that a certain genetic affinity of present-day Canary Islanders to Northwest African Berbers mainly stems from the autochthonous population rather than slaves captured on the neighbouring African coast. The slave trade after the European conquest left measurable, though minor, traces in the mtDNA pool of the Canary Islands, which in its majority testifies to the European immigration.

The aim of our study was to characterize the tachykinin receptor population in the oestrogen-primed rat uterus. For this purpose, we investigated the receptor type(s) responsible for tachykinin-induced contraction of longitudinally-arranged smooth muscle layer. The effects of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and several of their analogues with well-defined selectivities for tachykinin NK1, NK2 and NK3 receptors were studied and their inhibition by the selective nonpeptide tachykinin receptor antagonists (S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR 140333, NK1-selective), (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR 48968, NK2-selective) and (R)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)- 4-phenylpiperidin- 4-yl)-N- methylacetamide (SR 142801, NK3-selective) was evaluated. Additionally, expression of tachykinin receptor mRNA was examined by using the reverse transcription-polymerase chain reaction (RT-PCR). SP, NKA, [Nle10]-NKA(4-10), the analogue with selectivity at the tachykinin NK2 receptor type, and NKB elicited concentration-dependent contractions of the rat uterus. The pD2 values were 5.95±0.19; 6.73±0.21; 7.53±0.12 and 5.76±0.21, respectively. The selective agonist for the tachykinin NK1 receptor [Sar9Met(O2)11]-SP produced a small phasic response in the nanomolar concentration range. The selective tachykinin NK3 receptor agonist [MePhe7]-NKB failed to induce any significant contraction. In the presence of the neutral endopeptidase inhibitor phosphoramidon (1 μM), the log concentration-response curves to exogenous tachykinins and their analogues were shifted significantly leftwards. The pD2 values were 6.12±0.10, 8.04±0.07, 7.89±0.03 and 6.59±0.07 for SP, NKA, [Nle10]-NKA(4-10) and NKB, respectively. In the presence of phosphoramidon (1 μM), [Sar9Met(O2)11]-SP (1 nM–0.3 μM) induced concentration-dependent contractions of increasing amplitude when only one concentration of drug was applied to each uterine strip and the pD2 value was 7.61±0.89. [MePhe7]-NKB induced small, inconsistent contractions and, therefore, a pD2 value could not be calculated. In experiments performed in the presence of phosphoramidon (1 μM), SR 48968 (3 nM–0.1 μM) caused parallel and rightward shifts in the log concentration-response curves of NKA. The calculated pKB value was 9.16±0.08 and the slope of the Schild regression was 1.28±0.24. SR 48968 (0.1 μM) also antagonized responses to SP with an apparent pKB value of 7.63±0.13. SR 48968 (0.1 μM) inhibited contractions elicited by NKB (1 nM–3 μM) and [Nle10]-NKA(4–10) (0.1 nM–3 μM) but had no effect on the response evoked by [Sar9Met(O2)11]-SP (0.1 μM). SR 140333 (0.1 μM) inhibited responses to SP with an apparent pKB value of 7.19±0.22. This compound did not significantly affect responses to NKA, [Nle10]-NKA(4-10) and NKB, but suppressed [Sar9Met(O2)11]-SP (0.1 μM)-induced contraction. SR 142801 (0.1 μM) had no effect on responses to natural tachykinins or their analogues. Total RNA was extracted from some of the uteri used in functional studies. RT-PCR assays revealed single bands corresponding to the expected product sizes encoding cDNA for tachykinin NK1 (587 base pairs) and NK2 receptors (491 base pairs) (n=6 different animals). A very low abundance transcript corresponding to the 325 base pairs product expected for the tachykinin NK3 receptor was detected. The present data show that functionally active tachykinin NK1 and NK2 receptors are expressed in the oestrogen-primed rat uterus. The NK2 receptor type seems to be the most important one involved in the contractile responses elicited by tachykinins. NK3 receptors are present in trace amounts and seem not to be involved in tachykinin-induced contractions. British Journal of Pharmacology (1998) 123, 259–268; doi:10.1038/sj.bjp.0701613

Alkaptonuria (AKU) is caused by lack of homogentisate 1, 2 dioxygenase (HGO) activity. From the complete sequence of a human HGO cDNA, primers were designed in order to obtain reverse transcription–polymerase chain reaction products from tissues with ectopic transcription amenable to diagnostic analysis. A search for mutations in HGO cDNA was performed in an AKU family using urine and blood samples. The results show complete cosegregation (Z = 6.32; θ = 0) between a CT transition at position 817 of the human HGO cDNA and AKU. This mutation predicts a ProSer replacement at amino acid 230, and generates an EcoRV site. Am. J. Med. Genet. 78:192–194, 1998. © 1998 Wiley-Liss, Inc.