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K Sabeur, AT Vo, and BA Ball

The aim of this study was to characterize angiotensin-converting enzyme (ACE) in canine testis. Detergent-extracted canine testes were sonicated in the presence of protease inhibitors and purified on an affinity column with the ACE inhibitor, lisinopril, as an affinity ligand for ACE. The fractions recovered were assessed for ACE enzyme activity via an enzyme kinetic microplate assay (at 330 nm) based on the hydrolysis of Fa-Phe-Gly-Gly (FAPGG) at pH 7.5 during an 8 min incubation. The specific activity of ACE in the starting testicular extracts was 3.53 +/- 0.99 mU mg(-1) protein with a 1588 times enrichment in ACE activity after lisinopril affinity chromatography (4239 +/- 2600 mU mg(-1) protein). The recovery efficiency of ACE after lisinopril affinity chromatography was 71.2%. The ACE activity in the detergent extracts and the purified fractions was inhibited significantly by 10 micromol captopril l(-1), a specific ACE inhibitor, and was restored to 88% of normal activity by the addition of the thiol-alkylating agent N-ethylmaleimide (0.5 mmol l(-1)) in the detergent extracts and the purified fractions incubated with captopril. The treatment of testicular extracts with 10 mmol EDTA l(-1) reduced the ACE activity significantly (5.40 +/- 1.26 versus 0.58 +/- 0.23 mU mg(-1)). The ACE activity was restored fully in the presence of zinc (5.28 +/- 0.70 mU mg(-1)). The anti-ACE antibody (raised against a 70 kDa protein from the periacrosomal plasma membrane of equine spermatozoa) recognized a 65-70 kDa protein in the detergent-extracted testes as well as in the affinity-purified fractions. This antibody also recognized a protein of similar molecular mass in ejaculated spermatozoa. ACE was localized in the periacrosomal area of the ejaculated spermatozoa and in spermatids in the seminiferous tubules. The results of this study demonstrate that ACE is present in canine testis and retains its enzyme activity after purification with lisinopril affinity chromatography. Activity of canine ACE is inhibited by captopril and EDTA and is restored in the presence of N-ethylmaleimide and zinc.

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K Sabeur, AT Vo, and BA Ball

Angiotensin II is a hormone with a wide array of physiological effects that exerts its effect via interaction with two major subtypes of receptor. The results of this study show that angiotensin II (from 1 to 100 nmol l(-1)) initiates acrosomal exocytosis in equine spermatozoa that have undergone capacitation in vitro in a TALP-TEST (Tyrode's albumin lactate pyruvate; 188.7 mmol TES l(-1), 84.8 mmol Tris l(-1)) buffer with cAMP. The acrosome reaction and sperm viability were assessed with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and Hoechst 33258, respectively. The initiation of the acrosome reaction by angiotensin II was strongly inhibited by losartan, a specific angiotensin II type 1 receptor antagonist. Although angiotensin II as well as progesterone both initiated the acrosome reaction in equine spermatozoa, there was no synergistic effect when both agonists were added simultaneously. Initiation of acrosomal exocytosis by angiotensin II was accompanied by a rapid and transient calcium influx that was assessed in capacitated spermatozoa loaded with Fura-2AM. In addition, the angiotensin II-mediated calcium influx was inhibited when spermatozoa were preincubated with losartan. Western blotting with an antibody against angiotensin II type 1 receptor detected a major sperm protein of 60 kDa. Indirect immunofluorescence of non-capacitated spermatozoa with the angiotensin II type 1 receptor antibody revealed labelling in the midpiece and tail. In capacitated spermatozoa, the angiotensin II type 1 receptor was localized mainly over the anterior region of the sperm head, the equatorial segment and occasionally on the postacrosomal region in addition to the sperm tail. In conclusion, this study demonstrated the ability of angiotensin II to stimulate the acrosome reaction in capacitated equine spermatozoa. This effect is mediated via the angiotensin II type 1 receptor and is accompanied by an increase in intracellular calcium.

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K Sabeur and B A Ball

Reactive oxygen species (ROS) play an important role in normal sperm function, and spermatozoa possess specific mechanisms for ROS generation via an NAD(P)H-dependent oxidase. The aim of this study was to identify the presence of an NADPH oxidase 5 (NOX5) in equine testis and spermatozoa. The mRNA of NOX5 was expressed in equine testis as detected by northern blot probed with human NOX5 cDNA and by RT-PCR. Immunoblotting with affinity purified α-NOX5 revealed one major protein in equine testis and other tissues. Immunolocalization of NOX5 showed labeling over the rostral sperm head with some labeling in the equatorial and post-acrosomal regions. In the testis, there was abundant staining in the adluminal region of the seminiferous tubules associated with round and elongating spermatids. The RT-PCR and sequence analysis revealed a high homology with human NOX5. This study demonstrates that NOX5 is present in equine spermatozoa and testes and therefore represents a potential mechanism for ROS generation in equine spermatozoa.

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K Sabeur, BA Ball, TM Nett, HH Ball, and IK Liu

This study evaluated the effect of a GnRH analogue conjugated to the cytotoxin, pokeweed antiviral protein (PAP), on reproductive function in adult, male dogs. Four dogs received 0.0042 mg GnRH-PAP kg(-1) hourly for 36 h, and four other dogs received 0.1 mg GnRH-PAP kg(-1) as one bolus injection daily for three consecutive days. One dog received a single bolus (0.1 mg x kg(-1)). Three adult male dogs received GnRH without the PAP conjugate, as controls. Twenty-five weeks after the initial treatment, all treated dogs received 0.1 mg GnRH-PAP kg(-1) as a single administration, whereas dogs in the control group received 0.0045 mg kg(-1) of the GnRH analogue. Serum concentrations of testosterone and LH were determined by radioimmunoassay, and testis size was measured for 9 months after treatment. Stimulation tests (5 microg GnRH kg(-1)) were used to evaluate LH release (-15, 0, 30, 60, 90, 120 min), which was assessed by measuring area under the curve. Serum testosterone concentrations were significantly lower (P<0.05) after treatment in the bolus and hourly groups than in the control group. Testosterone concentrations fell to less than 50 pg x ml(-1) in three of four dogs in the bolus group and one of four dogs in the hourly group by week 8-9 after treatment. Basal LH was lower (P<0.05) in the bolus and hourly groups than in the control group between weeks 0 and 33 after treatment. Treatment with GnRH-PAP reduced (P<0.05) LH release after GnRH stimulation in the bolus and hourly groups compared with the control group. Testis volume was lower (P<0.05) in all treated versus control dogs. In conclusion, administration of the conjugate GnRH-PAP at a 25 week interval resulted in a major disruption of reproductive parameters in male dogs; this effect was maintained for 11-12 weeks after a second injection of GnRH-PAP.

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K. M. Henderson, L. C. Savage, R. L. Ellen, K. Ball, and K. P. McNatty

Summary. Romney ewes were infused with ovine FSH (NIADDK-oFSH-16) for 48 h from the initiation of luteolysis with cloprostenol. Doses of 2·5 or 5 μg/h which partly or completely prevented the normal preovulatory decline in plasma FSH concentrations caused a significant increase in mean ovulation rates. Ovulation rates were not increased significantly if the FSH (5 μg/h) was infused for only 20 h starting from the initiation of luteolysis or 24 h later. Infusion of a less potent and relatively impure preparation of FSH (i.e. FSH-P) at 0·5 mg/h for 48 h after cloprostenol treatment also increased the mean ovulation rate significantly. However, if the FSH-P was given for only the first 24 h, or if the start of the infusion was delayed for more than 12 h, mean ovulation rates were not increased significantly. Infusion of LH (NIADDK-oLH-25, 5 μg/h) for 48 h from the initiation of luteolysis decreased the mean ovulation rate significantly.

Administration of bovine follicular fluid to suppress plasma FSH concentrations below normal during the first 24 h after cloprostenol injection did not delay oestrus. However, oestrus was delayed by ∼2 days if plasma FSH concentrations were reduced by bovine follicular fluid 24 h after the initiation of luteolysis.

As ovulation rate increased, the mean weight of individual corpora lutea of each ewe decreased. In ewes with a single ovulation, most corpora lutea weighed > 600 mg, but as the ovulation rate increased the proportion of corpora lutea present weighing < 400 mg rose steadily. The mean concentrations of luteal tissue progesterone were not influenced significantly by ovulation rate.

Keywords: sheep; ovulation rate; FSH; follicular fluid

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K. P. McNatty, D. A. Heath, N. L. Hudson, K. Ball, and L. Condell

Summary. No gene-specific differences were found during either the luteal or follicular phases of the oestrous cycle in the venous secretion rates of ovaries or in concentrations of immunoreactive inhibin in peripheral plasma between Booroola ewes that were homozygous carriers (BB) or non-carriers (++) of the Fec B gene. In three experiments in which concentrations of plasma inhibin and follicle-stimulating hormone (FSH) were compared, gene-specific differences were noted for FSH (P < 0·05), but no significant correlations were noted between FSH and inhibin for either genotype. Granulosa cells and follicular fluid, but not theca interna, stroma or corpora lutea, were the major intra-ovarian sites of inhibin; no gene-specific differences were noted for inhibin concentrations in follicular fluid or in any of the intra-ovarian tissues. The mean concentrations of inhibin in follicular fluid remained constant irrespective of follicular diameter whereas the mean total contents of inhibin increased significantly with increasing diameter (P < 0·05). Inhibin secretion rates were four times higher in ovaries with oestrogen-enriched follicles (i.e. ≥ 50 ng oestradiol ml−1) than in ovaries with no such follicles (P < 0·01). Moreover, inhibin concentrations were higher in follicular fluid of oestrogen-enriched follicles than in those with low oestrogen (i.e. < 50 ng ml−1; P < 0·05). Ovariectomy resulted in a significant reduction in concentrations of immunoreactive inhibin from plasma (P < 0·01). The residual plasma inhibin in some Booroola ewes was not associated with genotype.

It is concluded that, although antral follicles are a major source of inhibin in Booroola ewes, immunoreactive inhibin is not associated with the Fec B gene and is not responsible for the gene-specific differences in concentrations of FSH in plasma.

Keywords: Booroola ewes; Fec B gene; inhibin; FSH; peripheral plasma; ovarian secretion rates

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K. P. McNatty, Katherine Ball, Marion Gibb, Norma Hudson, and D. C. Thurley

Summary. Anoestrous ewes (N = 3) were treated with a 500 ng GnRH pulse administered via a jugular cannula every 2 h for 40 to 80 days. Plasma concentrations and therefore presumed ovarian activity changed cyclically with each progestational cycle (n = 10) lasting 14·0–18·5 days. It is concluded that, by increasing the frequency of GnRH secretory episodes from an apparent endogenous level of one episode per 3·6 h to at least one every 2·0 h, cyclic ovarian activity can be restored to seasonally anoestrous sheep.

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K. P. McNatty, N. Hudson, M. Gibb, K. Ball, K. M. Henderson, D. A. Heath, S. Lun, and L. E. Kieboom

Summary. Injection of steroid-free bovine follicular fluid (bFF; 2 × 5 ml s.c. 12h apart) into anoestrous ewes lowered plasma FSH concentrations by 70% and after 24 h had significantly (P < 0·01) reduced the number of non-atretic follicles (≥ 1 mm diam.) without influencing the total number of follicles (≥ 1 mm diam.) compared to untreated controls. Hourly injections of FSH (10 μg i.v. NIH-FSH-S12) for 24 h did not influence the number of non-atretic follicles but did negate the inhibitory effects of bFF on follicular viability. Hourly injections of FSH (50 μg i.v., NIH-FSH-S12) + bFF treatment for 24 h significantly increased the total number of non-atretic follicles, and particularly the number of medium to large non-atretic follicles (≥ 3 mm diam.) compared to the untreated controls (both P < 0·01). The 10μg FSH regimen (without bFF) significantly increased aromatase activity in granulosa cells from large ( ≥ 5 mm diam.; P < 0·01) but not medium (3–4·5 mm diam.) or small (1–2·5 mm diam.) follicles compared to controls. The 10 μg FSH + bFF regimen had no effect on granulosa-cell aromatase activity compared to the controls. However, the 50 μg FSH plus bFF regimen increased the aromatase activity of granulosa cells from large, medium and small non-atretic follicles 2·6-, 8·3- and ≥ 11-fold respectively compared to that in the control cells.

Ewes (N = 11) that ovulated 2 follicles had significantly higher plasma FSH concentrations from 48 to 24 h and 24 to 0 h before the onset of a cloprostenol-induced follicular phase (both P < 0·01) than in the ewes (N = 12) that subsequently ovulated one follicle. Hourly FSH treatment (1·6 μg i.v., NIAMDD-FSH-S15) for 24 h but not for any 6 h intervals between 48 and 24 h or 24 and 0 h before a cloprostenol-induced luteolysis also resulted in significant increases (P < 0·05) in the number of ewes with 2 ovulations.

We conclude that (1) the number of non-atretic antral follicles in sheep ovaries is influenced by plasma FSH concentrations; (2) the level of follicular oestradiol biosynthesis can be enhanced by FSH treatment; and (3) sustained elevations of plasma FSH concentrations for 24 h but not 6 h within 48 h of the onset of luteolysis significantly enhances the ovulation rate in Romney ewes.

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K. P. McNatty, M. Fisher, F. Collins, N. L. Hudson, D. A. Heath, K. Ball, and K. M. Henderson

Summary. During 12 sampling days before ovariectomy the mean plasma FSH but not LH concentrations in FF ewes were higher (P < 0·01) than those in ++ ewes (16 ewes/genotype). After ovariectomy increases in the concentrations of FSH and LH were noted for ewes of both genotypes within 3–4 h and the rates of increase of FSH and LH were 0·18 ng ml−1h−1 and 0·09 ng ml−1 h−1 respectively for the first 15 h. From Days 1 to 12 after ovariectomy, the overall mean ± s.e.m. concentrations for FSH in the FF and ++ ewes were 8·1 ± 0·6 and 7·1 ± 0·4 ng/ml respectively and for LH they were 2·7 ± 0·3 and 2·1 ± 0·2 ng/ml: these differences were not statistically significant (P = 0·09 for both FSH and LH; Student's t test). However, when the frequencies of high FSH or LH values after ovariectomy were compared with respect to genotype over time, significant F gene-specific differences were noted (P < 0·01 for both FSH and LH; median test).

In Exp. 2 another 21 ewes/genotype were blood sampled every 2nd day from Days 2 to 60 after ovariectomy and the plasma concentrations of FSH and LH were more frequently higher in FF than in ++ ewes (P < 0·01 for FSH and LH). The F gene-specific differences in LH concentration, observed at 21–36 days after ovariectomy were due to higher mean LH amplitudes (P < 0·025) but not LH peak frequency in FF than in ++ ewes.

Collectively the evidence shows that the greater frequency of high plasma gonadotrophin concentrations in FF compared to ++ Booroola ewes occurs independently of ovarian hormones and that the principal site(s) of F gene expression may not be in the gonad.

Keywords: Booroola ewes; ovariectomy; plasma FSH; plasma LH

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K. P. McNatty, N. Hudson, K. M. Henderson, M. Gibb, L. Morrison, K. Ball, and P. Smith

Summary. The mean plasma concentrations of FSH and LH were significantly higher in FF ewes than in ++ ewes with those for F+ animals being consistently in between. These gene-specific differences were found during anoestrus, the luteal phase and during a cloprostenol-induced follicular phase, suggesting that the ovaries of ewes with the F-gene are more often exposed to elevated concentrations of FSH and LH than are the ovaries of ewes without the gene.

The gene-specific differences in LH secretion arose because the mean LH amplitudes were 2–3 times greater in FF compared to ++ ewes with the LH amplitudes for F + ewes being in between. The LH pulse frequencies were similar. In these studies the pulsatile nature of FSH secretion was not defined.

The pituitary contents of LH during the luteal phase, were similar in all genotypes whereas for FSH they were significantly higher in the F-gene carriers compared to ++ ewes. The pituitary sensitivity to exogenous GnRH (0·1, 0·5, 5·0 and 25 μg i.v.) was related to genotype. Overall the LH responses to GnRH were lower in FF ewes than in ++ ewes with the results for the F+ ewes being in between. The FSH responses to all GnRH doses in the FF genotype were minimal (i.e. < 2-fold). In the other genotypes a > 2-fold response was noted only at the highest GnRH dose (i.e. 25 μg). Treatment of FF and F+ but not ++ ewes with GnRH eventually led to a reduced FSH output, suggesting that the pituitary responses to endogenous GnRH were being down-regulated in the F-gene carriers whereas this was not the case in the non-carriers.

Collectively these data confirm that peripheral plasma and the pituitary together with the ovary are compartments in which F-gene differences can be observed. In conclusion, these findings raise the possibility that F-gene-specific differences may also extend to the hypothalamus and/or other regions of the brain.