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D. A. SCHELLPFEFFER and A. G. HUNTER

Summary.

The origins of the major proteins of boar seminal plasma were identified using starch gel electrophoresis, gel filtration on Sephadex G-200 and surgical removal of various accessory sex glands.

Seminal vesicular secretion accounted for all the major proteins in seminal plasma which migrated to the cathode. Bulbo-urethral proteins could not be detected electrophoretically or by gel filtration. Prostatic-urethral fluid contained at least one protein which migrated to the anode and was not of serum origin. Secretions from the epididymis and/or testis contained at least two proteins which migrated to the anode that were eventually voided in the seminal plasma of the ejaculate.

Boar seminal plasma separated on Sephadex G-200 into three major peaks corresponding to molecular weights of approximately 155,000 (Peak B), 55,000 (Peak A) and 34,000 (Peak C). Peak A proteins were primarily of seminal vesicular origin, while Peak B proteins were primarily of epididymal or testicular origin. Peak C consisted of proteins from all regions of the reproductive tract. Haemagglutinating activity was located between Peaks A and B, associated with molecules of approximately 68,000 molecular weight, and was absent after removal of the seminal vesicles.

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A. G. HUNTER and H. D. HAFS

Summary.

Bombardment of bovine spermatozoa with glass beads and subsequent extraction in phosphate buffered sodium chloride yielded soluble sperm-specific proteins (approximately 10% yield) sufficient for physicochemical analysis.

Moving boundary electrophoresis revealed three sperm-specific components with mobilities of 2·0, 3·8 and 5·1×10-5 cm2 volt-1 sec-1 at pH 8·6 in barbital (μ = 0·10). The proportions of these components were 20, 50 and 30%, respectively. Sedimentation velocity ultracentrifugal analysis revealed at least three sedimentation gradients. The two major gradients had S20 values of 1·7 and 12·6. The third exhibited polydispersion. Diffusion coefficients of 4·2 and 10·2×10-7 cm2 sec-1 were calculated for two sperm-specific antigens by an agar-gel-diffusion method.

Three protein fractions were isolated by chromatographic elution from diethylaminoethyl (deae) cellulose with an ionic gradient. The first protein fraction eluted was not bound to the deae cellulose. It migrated toward the cathode in agar-gel electrophoresis at pH 8·4, and was firmly bound to carboxymethylcellulose at pH 6·0, 7·6 and 11·0. A second protein fraction from the deae cellulose appeared just after the ionic gradient commenced, and a third protein fraction appeared when the ionic gradient approached 0·2 m-sodium chloride.

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A. P. Beard and M. G. Hunter

The role of oestradiol in the control of premature luteolysis (previously shown to occur by the normal luteolytic mechanism involving PGF and oxytocin) was investigated in anoestrous ewes induced to ovulate using GnRH (250 ng every 2 h for 24 h followed by 125 μg on day 0) without progesterone pretreatment. Seven ewes were administered charcoal stripped bovine follicular fluid (bFF) on days 1–5 (2 ml by s.c. injection every 8 h) together with oestradiol on days 2–4 (4 μg in 1 ml of corn oil by i.m. injection every 8 h). Ten ewes were treated with bFF and corn oil (as above), and ten ewes received saline and corn oil (control group). All ewes were treated with 1 μg oxytocin (i.v.) on day 4 and plasma was collected for measurement of 13,14-dihydro-15-keto PGF (PGFM). Blood samples were collected for measurement of progesterone and oestradiol (day−2 to 15). The ewes in the control group that responded to GnRH formed either normal (50% of ewes) or short-lived (50% of ewes) corpora lutea identified by progesterone profiles. The proportion of ewes that displayed premature luteolysis was reduced (P < 0.05) by bFF treatment alone (to 11% of ewes), and increased (P < 0.001) by bFF plus oestradiol treatment (to 100%). bFF treatment suppressed oestradiol concentrations (P < 0.01), whereas bFF plus oestradiol treatment increased oestradiol concentrations (P < 0.001) on days 1–5. The high oestradiol concentrations appeared to stimulate the premature luteolytic mechanism as the mean PGFM response to oxytocin was higher in the ewes treated with bFF plus oestradiol than in the other two groups (P < 0.001). In addition, the control ewes that formed short-lived corpora lutea had higher oestradiol concentrations (days 1–5) than did ewes with normal corpora lutea (P = 0.05). This study suggests that short-lifespan corpora lutea are the result of increased oestrogenic stimulation of the luteolytic mechanism during the early luteal phase (following a lack of prior exposure to progesterone) which can be overcome by suppressing oestradiol secretion. This finding demonstrates that oestradiol plays a key role in the initiation of premature luteolysis, probably through stimulation of the prostaglandin response to oxytocin.

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A. G. HUNTER and H. D. HAFS

Summary.

The antigenicity and cross-reactions of bovine seminal constituents were studied using complement fixation, agar-gel-diffusion, and immuno-electrophoresis. Bovine ejaculated spermatozoa, seminal plasma, and blood serum contained common antigens such that antisera produced with one also reacted with the other two. Spermatozoa per se were antigenic when injected intradermally with Freund's adjuvant into rabbits. In addition, ejaculated spermatozoa were coated with seminal plasma proteins. At least some of these coating antigens were found on spermatozoa from the vas deferens and must therefore have been deposited before the contributions of the major accessory sex glands came in contact with the spermatozoa. Antisera prepared against epididymal spermatozoa reacted with epididymal and ejaculated spermatozoa but not with seminal plasma or blood serum.

Ejaculated spermatozoa possessed at least seven antigens. At least five were shared with seminal plasma and at least one with blood serum. Absorption of anti-sperm immune sera with seminal plasma and blood serum revealed that epididymal and ejaculated spermatozoa shared at least three protein antigens which were not found in seminal plasma or blood serum.

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A. G. HUNTER and H. O. NORNES

Summary.

The relationship between the sperm-coating antigens of rabbit seminal plasma and the phenomenon of decapacitation was studied using agar-gel diffusion, immuno-electrophoresis, chromatography on Sephadex G-200, and polyacrylamide vertical gel electrophoresis.

Interpretation of data obtained with these techniques led to the conclusion that a sperm-coating antigen of seminal plasma origin possessed biological activity for blocking fertilization. The sperm-coating antigen was a glycoprotein of approximately 170,000 molecular weight, migrated in an electric field similar to a serum slow β-globulin and was still present in the seminal fluid of vasectomized males. This sperm-coating antigen was absent from the inactive upper supernatant fluid fraction of seminal plasma after 4 hr of ultracentrifugation at 105,000 g and was present in the active ultracentrifugal pellet.

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A. P. Beard and M. G. Hunter

A steroid-treated ovariectomized ewe model was used to investigate the role of progesterone pretreatment in the control of functional oxytocin receptor concentrations during the early luteal phase. Ovariectomized ewes (n = 28) were injected with oestradiol for 2 days (final injection = day 0) with or without progesterone pretreatment (progestagen sponge for 10 days). Ewes were then given high or low concentrations of progesterone combined with high, low or zero concentrations of oestradiol in a pattern known to simulate the early luteal phase profile (n = 4 per group). Ewes were given 1 μg oxytocin (i.v.) on day 4 and plasma was collected to assay 13,14-dihydro-15-keto PGF. The concentration of progesterone and oestradiol administered had no effect on the concentration of 13,14-dihydro-15-keto PGF following oxytocin administration (P > 0.05). However, the group that was not pretreated exhibited a small but significant 13,14-dihydro-15-keto PGF response in comparison with the equivalent pretreated group (P < 0.05). In a subsequent study, ewes were divided into groups pretreated and not pretreated with progesterone; both groups were given oestrous concentrations of oestradiol and high concentrations of progesterone and oestradiol together. On day 0, 2, 3 or 4, ewes from each group (n = 3, 3, 4 and 4, respectively) were given 1 μg of oxytocin i.v., and the endometrium was collected to measure the binding of oxytocin receptors. Oxytocin caused a significant (P < 0.05) increase in the concentration of 13,14-dihydro-15-keto PGF in all ewes on day 0 but not on days 2, 3 or 4. Oxytocin receptor concentrations were maximal on day 0 and basal by day 4. The decline in receptor concentrations occurred more rapidly in the progesterone-pretreated than in the ewes that were not pretreated. This study has shown that progesterone pretreatment alters the subsequent steroid hormone control of oxytocin receptor concentrations, and has identified the delayed decline in oxytocin receptor concentrations as the potential cause of premature luteolysis in ewes that are not pretreated.

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A. P. Beard and M. G. Hunter

Two experiments investigate the effects of oxytocin and progesterone on premature luteolysis in ewes. In Expt 1, 20 anoestrous ewes were induced to ovulate by multiple injections of GnRH (250 ng i.v. every 2 h for 24 h) followed by a bolus injection of GnRH (125 μg, i.v.). Ten ewes received a continuous infusion of oxytocin from the day after the GnRH bolus injection and the other ten ewes were infused with saline. Oxytocin infusion had no significant effect on the proportion of ewes with short luteal phases (P > 0.05). All ewes that had luteal phases of normal duration from either group (n = 9) exhibited a transient increase in plasma concentrations of progesterone 2 h after insertion of the pump. In Expt 2, 25 anoestrous ewes were treated with GnRH as in Expt 1. Five ewes were pretreated with progestagen for 11 days and ten ewes received progesterone (12 mg, i.m.) 24 h after the bolus injection of GnRH. All animals received an oxytocin injection (1 μg, i.v.) on day 4 after the GnRH bolus. All five ewes that were pretreated with progestagen had normal luteal function and none exhibited a 13,14-dihydro-15-keto PGF (PGFM) response to oxytocin. None of the ten ewes injected with progesterone had a normal luteal phase and six ewes exhibited a PGFM response to oxytocin. Four ewes in the control group had normal luteal function and three had short luteal phases. It is concluded that (1) administration of oxytocin from about the time of ovulation does not prevent premature luteal regression; (2) a transient increase in progesterone at about the time of ovulation is associated with luteal phases of normal duration; (3) a more extended exposure to progesterone at about the time of ovulation prevents normal luteal function and may inhibit luteinization and (4) pretreatment with progesterone prevents luteolysis by reducing the uterine response to oxytocin early in the luteal phase.

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G. L. HUNTER and A. W. LISHMAN

Summary.

By means of ovarian examination and teasing with vasectomized rams, the intervals to first post-partum ovulation and oestrus were determined in a 23-factorial experiment with twenty-four German Merino ewes which lambed between 28th October and 15th November 1966. First ovulation occurred 32·1 ± 1·9 (8 to 45) days after lambing. Three, fifteen and six ewes had none, one or more than three silent ovulations respectively, before showing heat after a mean interval from lambing for all ewes of 57·1±4·5 days. The regression of post-partum interval to ovulation (Y) on ewe's weight 3 days post partum (X) was significant (P<0·01) ; for ewes weighing 83 to 162 lb, Y = 55·15–0·21X. The regression of post-partum interval to oestrus on ewe's weight was not significant, and neither were the effects of treatments, namely, weaning at 3 or 20 days post partum, supplementary feeding of 0 or 1 lb maize grain daily from 3 to 28 days post partum, and joining with vasectomized rams at 3 or 14 days post partum.

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M. G. Hunter, S. A. Grant and G. R. Foxcroft

Summary. Ovaries were collected from naturally cycling gilts during the preovulatory period and the stage relative to the LH surge estimated by measurement of oestradiol and progesterone concentrations in follicular fluid. Many of the follicles recovered had become flaccid with an associated increase in follicular fluid viscosity. Marked infolding of both the granulosa and theca tissue in some follicles suggested early luteinization. However, these morphological changes did not necessarily occur simultaneously in the same follicle, or in all follicles within an ovary. Moreover, they were not consistently related to characteristic differences in the concentration of follicular fluid steroids, suggesting either that the morphological and biochemical aspects of the luteinization of follicles may be independently controlled, or may respond at different rates to the same signal.

Keywords: pig; follicle; histology; heterogeneity

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S. A. Grant, M. G. Hunter and G. R. Foxcroft

Summary. Ovaries were recovered from groups of naturally cyclic pigs (N = 5) on each of Days 16, 18, 20 and 21 of the oestrous cycle. Follicular diameter, follicular fluid volume and concentrations of oestradiol, testosterone and progesterone, and granulosa cell number were determined in all follicles ⩾2 mm in diameter (n = 511). In alternate follicles either granulosa cell aromatase activity and theca testosterone content or 125I-labelled hCG binding to granulosa and theca were determined. The mean total number of follicles recovered per animal decreased as the follicular phase progressed and a strong positive relationship (P < 0·001) existed between follicular diameter and volume on all days. The number of granulosa cells recovered per follicle was variable, and not related to oestrogenic activity of the follicles. Mean follicular fluid oestradiol, testosterone and 125I-labelled hCG binding all increased until Day 20 and decreased on Day 21, whereas mean theca testosterone content, 125I-labelled hCG binding to theca tissue and aromatase were all maximal on Day 21. On Days 20 and 21 a subset of 14–16 large follicles was readily distinguishable from the remaining smaller, less oestrogenically active population in each animal. Yet, consistently within these subsets there was a difference in follicular diameter of ∼ 2·0 mm and also a considerable range of biochemical development even among follicles of equal size. These results indicate asynchrony at the time of recruitment and selection among follicles destined to ovulate and suggest that heterogeneity continues into the immediate preovulatory period.

Keywords: follicle; development; pig; heterogeneity