Functional and biochemical data have suggested a role for the cytochrome P450 arachidonate monooxygenases in the pathophysiology of hypertension, a leading cause of cardiovascular, cerebral, and renal morbidity and mortality. We show here that disruption of the murine cytochrome P450, family 4, subfamily a, polypeptide 10 (Cyp4a10) gene causes a type of hypertension that is, like most human hypertension, dietary salt sensitive. Cyp4a10 -/-mice fed low-salt diets were normotensive but became hypertensive when fed normal or high-salt diets. Hypertensive Cyp4a10 -/-mice had a dysfunctional kidney epithelial sodium channel and became normotensive when administered amiloride, a selective inhibitor of this sodium channel. These studies (a) establish a physiological role for the arachidonate monooxygenases in renal sodium reabsorption and blood pressure regulation, (b) demonstrate that a dysfunctional Cyp4a10 gene causes alterations in the gating activity of the kidney epithelial sodium channel, and (c) identify a conceptually novel approach for studies of the molecular basis of human hypertension. It is expected that these results could lead to new strategies for the early diagnosis and clinical management of this devastating disease. IntroductionPrevalence, complexity, and multiple medical and socioeconomic consequences make hypertension a major health challenge for most of the Western world (1). While environmental factors and coexist ing conditions play a role in the development and progression of hypertension, segregation and linkage analyses indicate that mul tiple genetic factors contribute to its complex etiology (2-7). Fur thermore, clinical studies show that the cardiovascular and renal morbidity and mortality resulting from hypertension are markedly reduced by timely diagnosis and early clinical intervention (1). As the kidneys play a central role in the control of body salt and fluid balance, they are frequent targets for the treatment of hypertension, especially those forms sensitive to dietary salt (2-5). However, since the molecular basis of prevalent forms of the disease remains uncer tain, its early diagnosis and treatment are largely symptomatic. It is expected that the identification of novel pathways/genes involved in blood pressure variations (3, 6, 7) will lead to new therapeutic targets and to improved diagnosis and prevention. Indeed, early detection and treatment are urgently needed to prevent the dangerous and profound consequences of untreated chronic hypertension.The metabolism of endogenous arachidonic acid (AA) to epoxy eicosatrienoic acids (EETs) and 20hydroxyeicosatetraenoic acid

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality, and epidemiological evidence suggests a role for sex-dependent mechanisms in the pathophysiology of hypertension. We show here that treatment of rats with 5␣-dihydrotestosterone increases the activity of the kidney arachidonate /-1 hydroxylase and the biosynthesis of 20-HETE (165 and 177% of control untreated male and female rats, respectively) and raises the systolic blood pressures of male and females rats by 46 and 57 mmHg, respectively. These androgen effects are associated with an upregulation in the kidney levels of CYP 4A8 mRNA and a decrease in CYP 4A1 transcripts. Dissected renal microvessels, the target tissue for most of the prohypertensive actions of 20-HETE, show an androgen-dependent upregulation of vascular CYP 4A8 mRNA and a fourfold increase in 20-HETE synthase activity. We propose that androgens regulate renal function and systemic blood pressure through a combination of transcriptional and hemodynamic mechanisms that are ultimately responsible for the regulation of renovascular tone and function. P-450 eicosanoids; androgens; CYP 4A8A NOW CONSIDERABLE BODY of evidence shows that microsomal P-450s (CYPs) participate in the in vivo metabolism of arachidonic acid (AA) and that products of this P-450-catalyzed pathway are in vitro modulators of renal ion transport and vascular reactivity and may be involved in the regulation of systemic blood pressure (3, 4,22,29). During catalytic turnover, the P-450 monooxygenase(s) metabolize AA via /-1 hydroxylation (AA /-1 hydroxylase) to 19-and 20-HETE and/or epoxidation (AA epoxygenase) to 5,8,11,or 14,5). Recombinant CYPs 4A1, 4A2, 4A8, and to a lesser extent 4A3, support the hydroxylation of AA to either 20-HETE or to mixtures of 16,19,31). All four CYP 4A isoforms are expressed in the male rat kidney (4,16,19,31), with CYPs 4A1 and 4A2/4A3 characterized as the predominant microsomal AA /-1 hydroxylases (16). The transcriptional regulation of rat CYPs P-450 4A1 and 4A2/4A3 by the peroxisomal proliferator activated receptor-␣ (45), as well as the sexually dimorphic, testosterone-sensitive expression of rat CYPs 4A2 and 4A8 and of murine Cyp 4a12, has been reported (18,20,40,42).Studies with the spontaneously hypertensive-Wistar Kyoto (SHR/WKY) rat model of spontaneous hypertension, an extensively characterized model of genetic hypertension (29), suggest a role for renal CYP 4As in the pathophysiology of the disease (3, 29). Thus based on 1) temporal associations between the increases in blood pressure, renal CYP 4A expression and 20-HETE formation (3, 29), 2) experimental manipulations of CYP 4A activity and/or expression (29,32,43), and 3)

To clarify the sperm count required for fertilization by artificial intravaginal insemination (AIVI), twenty-nine female cats were examined. Six male cats aged 2-12 years with normal semen quality, copulation capability, and fertility were used. In AIVI, animals received administration of 250 iu hCG once or 100 iu twice on days 2-4 of estrus to induce ovulation, and were inseminated 15, 20, or 30 hr after the initial hCG administration. The success of ovulation was judged by elevation of the peripheral progesterone level after hCG administration. AIVI was investigated at three sperm counts, 20 x 10(6) (Experiment 1), 40 x 10(6) (Experiment 2), and 80 x 10(6) (Experiment 3), with semen collected by the artificial vagina method. Semen was infused in the vagina under general anesthesia by advancing a 9 cm-long nylon probe with 1.5 mm diameter connected to a 1 ml syringe in the vagina for 3-4 cm. Ovulation was induced in 43 of 45 animals (95.6%). One of 16 animals was fertilized (conception rate: 6.6%) by AIVI in Experiment 1. In Experiments 2 and 3, conception was obtained in six of 18 animals (33.3%) and seven of nine animals (77.8%), respectively, and the mean numbers of kits were 4.0 +/- 0.4 and 3.3 +/- 0.5, respectively, and the mean numbers of kits were 4.0 +/- 0.4 (SE) and 3.3 +/- 0.5, respectively, showing no significant difference. There were no differences in the time of insemination after hCG administration and the conception rate among these groups. Our findings showed that the number of sperm required for fertilization by AIVI of fresh semen in cats was 80 x 10(6).

ABSTRACT. The sperm count required were investigated to obtain a conception rate of 80% by unilateral intrauterine insemination (UIUI) of fresh semen in cats. The conception rates obtained by insemination before and after ovulation were also examined. Thirty-six female cats aged 1-7 years were used in the experiments, and the number of experimental cases was 44. Seven male cats aged 2-12 years from which semen could be collected by the artificial vagina method were used. In artificial insemination, 100 iu × 2 or 250 iu of hCG was administered on days 2-4 of estrus, and sperm were introduced into the uterine horn with a greater number of ovulations (or mature follicles) 15, 20 and 30 hr after hCG administration by laparotomy. The inseminated sperm counts were 2 × 10 6 (Exp. 1), 4 × 10 6 (Exp. 2), and 8 × 10 6 (Exp. 3). As a result, ovulation was induced in 42 of 44 cases (induction rate: 95.5%) regardless of the dosage of hCG. Conception was obtained by UIUI in two of 16 animals (conception rate: 12.5%) in the Exp. 1, five of 16 animals (31.3%) in Exp. 2, and eight of 10 animals (80.0%) in Exp. 3. Regarding the relationship between the ovulation state at insemination and conception, the conception rate obtained by insemination before ovulation was clearly higher than that obtained by insemination after ovulation (p<0.05). Regarding the number of kits compared to the number of ovulations on the inseminated side, the percentages of cases in which the number of kits exceeded the number of ovulations on the inseminated side were similar in all groups inseminated with a different number of sperm. It is therefore necessary to investigate conception rates obtained by bilateral insemination to increase the fertility rate. Based on the above findings, it was shown that the sperm count required for fertilization by UIUI is 8 × 10 6 . KEY WORDS: feline, fresh semen, intrauterine insemination.

ABSTRACT. Frozen feline semen was prepared using two types of extenders, egg yolk Tris-fructose citric acid (EYT-FC) and egg yolk sodium citrate solution (EYC), and the semen qualities after thawing and the conception rates obtained by unilateral intrauterine horn insemination (UIUI) were investigated. Cats used in the experiment were six males and 11 females aged 2-12 years (the number of experimental cases was 17). For preparation of frozen semen, semen collected by the artificial vagina method was adjusted to 1 × 10 8 sperm/ml and 7% glycerol, put in 250 µl straws, and then frozen using a cell freezer. The mean sperm motility after thawing was 30.0 ± 9.7 (SE) % in the semen prepared with EYT-FC and 30.0 ± 3.3% in the semen prepared with EYC. Four of seven animals were fertilized by UIUI using two straws in both extenders, and the conception rate was 57.1%. The mean ratios of number of kits to the number of ovulations in the inseminated side were 61.1 ± 24.5% and 30.5 ± 3.4% for EYT-FC and EYC, respectively, showing that the ratio tended to be higher in the semen prepared with EYT-FC. The above findings, comparing the two extenders for preparation of frozen feline semen, showed that EYT-FC is slightly superior to EYC. To increase conception and fertility rates, it may be important to increase the sperm count for insemination and to inseminate both uterine horns. KEY WORDS: feline, frozen semen, intrauterine insemination, straw method.J. Vet. Med. Sci. 62(12): 1247-1251 Feline artificial insemination (AI) using frozen semen has been reported only by Platz et al. in 1978 [3]. In their study, 50-100 × 10 6 viable sperms were inseminated into the vagina. Although only six of 56 animals were fertilized, obtaining a conception rate of 10.7%, these were the first cases of fertilization by frozen semen in cats. We previously performed intravaginal insemination (IVI) of fresh semen and clarified that a number of sperms, 80 × 10 6 , are required for fertilization [6]. This sperm count is equal to the number of sperm in two ejaculations in cats [5]. We also clarified the sperm count required for fertilization by unilateral intrauterine horn insemination (UIUI) of fresh semen to be 8 × 10 6 in cats, which is 1/10 of that by IVI [7]. Freezing markedly reduces motility and viability of sperms. Accordingly, it is important to develop a technique of fertilizaiton using a small number of sperm. Therefore, intrauterine insemination (IUI) may be appropriate for feline AI using frozen semen rather than IVI.The method using frozen feline semen reported by Platz et al. was the pellet method, which may pose difficulties for storage and use [3]. Therefore, we investigated freezing of feline semen by the straw method.Platz et al. used egg yolk lactose as the extender for frozen feline semen [3]. However, the semen qualities after thawing were not reported, and thus, we could not evaluate the extender used in their study. Therefore, we prepared frozen feline semen using egg yolk fructose citric acid (EYT-FC) [6,7], which we use as an e...