Summary. An evaluation of a method utilizing zona-free hamster ova to test the fertility of human spermatozoa has shown that (i) the induction of superovulation in immature animals provides the most convenient method of obtaining mature ova for study; (ii) motile spermatozoa are best prepared by the technique of layering; (iii) an 18 h incubation at 37°C (which is associated with capacitation) in an atmosphere of air (pH of medium 8·2) is preferable to one of 5% CO2 (pH of medium 7·2); (iv) the incubation and insemination densities of spermatozoa should be >1 × 106 and <10 × 106/ml; (v) spermatozoa do not remain motile, or capable of binding to or penetrating ova, after about 30 h in culture; and (vi) intra- and inter-assay variations are acceptable. The spermatozoa from 15 healthy men of proven fertility and 15 subfertile patients with normal spermiograms were evaluated for their ability to bind to and penetrate zona-free hamster ova. Of the 476 ova inseminated with spermatozoa from the fertile men > 5 spermatozoa/ovum consistently bound to the vitelline membrane and 284 ova 59·7%) had swollen sperm heads or pronuclei (still with tails attached) in their ooplasm. The range of individual penetration rates was 23·5–88·9%. Of the 586 ova tested with spermatozoa from the infertile subjects only 11 (1·9%) showed any evidence of penetration (range of individual penetration rates 0–8·7%) and binding to the vitelline membrane was poor (0 or <5 spermatozoa/ ovum). Spermatozoa from a further 9 infertile men who had abnormal spermiograms also gave poor penetration rates (4/300 ova, 1·3%). It is concluded that this bioassay has a useful role as an additional test to the classic spermiogram, but that its routine use is best reserved for selected cases of unexplained infertility.
J. P. P. Tyler, J. P. Pryor and W. P. Collins
J. P. P. Tyler, Janet Simpson and W. P. Collins
Summary. The androgenic status of female mice was assessed by measuring the concentration of testosterone-17β-glucuronide in serial samples of unextracted urine. The subcutaneous administration of testosterone (50 μg in lauric acid ethyl ester) resulted in a significant increase (P < 0·01) of the mean ± s.d. concentration of urinary testosterone-17β-glucuronide (25·9±6·5 to 71·7± 12·9 ng/ml) within 2 h. The 2-h values after the administration of 50 μg androstenedione or 50 μg dehydroepiandrosterone were 22·8 ±4·4 to 81·8 ± 7·8 ng/ml (P < 0·001) and 23·4 ± 2·7 to 121·8 ± 20·3 ng/ml (P < 0·001) respectively. The values decreased progressively over the next 24 h.
After the induction of superovulation with PMSG and hCG, the values for testosterone-17β-glucuronide increased significantly (P < 0·01) during the periovulatory period (5–24 h after hCG injection). The mean ± s.d. value in pregnancy was higher (61·8 ± 11·6 ng/ml; P < 0·001) than that in non-pregnant animals (30·7 ± 12·3 ng/ml) and remained relatively constant between Days 1 and 16.
P. L. Matson, J. P. P. Tyler and W. P. Collins
Summary. Follicular growth was stimulated by the administration of PMSG to immature female hamsters. Changes in the number and size of follicles, their content of progesterone, testosterone, oestradiol, and oestrone (determined in whole follicular homogenates) and the meiotic status of the oocytes, were followed for 4 days to monitor follicular development and ageing. The results showed that 92% of small antral follicles (371–590 μm) were seen 24–30 h after PMSG stimulation and that the maximum number of medium- (591–740 μm) and large- (>740 μm) sized antral follicles occurred >48 h later. There was a progressive increase in the concentrations of testosterone, oestradiol and oestrone over the first 48–54 h, but maximal values of oestrone (geometric mean (limits of 1 s.d.): 326·5 (230·4–462·7) pg/follicle) lagged 24 h behind the highest values for testosterone (352·5 (242·0–456·0) pg/follicle) and oestradiol (718·4 (315·7–1634·7) pg/follicle). The progesterone content of all follicles did not change significantly during the first 72–78 h (range of geometric mean values: 0·25–1·06 ng/follicle), but at 96–102 h two populations of mediumand large-sized follicles could be distinguished on the basis of a low progesterone concentration (Group 5a: medium, 0·27 (0·13–0·55) ng/follicle; large, 0·1 (0·08–0·13 ng/follicle; P < 0·01) or a high concentration (Group 5b: medium, 7·77 (3·12–19·36) ng/follicle: large, 13·10 (8·68–19·76 ng/follicle; P < 0·01) and this grouping corresponded to the peripheral plasma progesterone values of the animals (Group 5a: 1·32 (0·51–3·42) ng/ml; Group 5b: 6·76 (5·05–9·05) ng/ml; P < 0·01). Differences in concentrations of testosterone, oestradiol and oestrone were not found in these two groupings at 96–102 h, but large antral follicles at this time contained more oestrone (x 1·5) than medium-sized follicles. Oocytes recovered from follicles up to 78 h after PMSG stimulation showed few signs of atresia (>80% viable) and had not resumed meiosis (>93% had a visible germinal vesicle). At 96–102 h oocytes from medium and large follicles with a low progesterone content maintained their viability (>88%), but 41% of oocytes recovered from medium-sized follicles showed evidence of germinal vesicle breakdown. However, in follicles containing high concentrations of progesterone the incidence of atresia was high (38–48%) and most oocytes had resumed meiosis (>97%).
A. C. MENGE, W. J. TYLER and L. E. CASIDA
Rabbit and bull spermatozoa were observed undergoing phagocytosis by polymorphonuclear leucocytes after being injected into ligated uteri of oestrous and luteal phase rabbits. An interaction between the species of spermatozoa injected and the reproductive phase of the rabbit occurred in the number of recovered spermatozoa.
Killing the bull spermatozoa prior to injection decreased the recovery rate significantly while killing the rabbit spermatozoa had no significant effect.
Treating either bull or rabbit spermatozoa with uterine exudates induced by bull spermatozoa decreased significantly the recovery rates from oestrous rabbits as compared to treatment with saline solution or exudates induced by rabbit spermatozoa.
In leucopenic rabbits, differences in recovery rates due to species of spermatozoa injected and reproductive phase of the rabbit were not evident. Approximately 75 to 80% of the spermatozoa injected were recovered compared to less than 20% for the controls.
In none of the experiments did the number of recovered leucocytes vary with the different treatments, except after the induction of leucopenia.
A. C. MENGE, W. H. STONE, W. J. TYLER and L. E. CASIDA
Immune sera produced against bull semen in cattle and against washed bull spermatozoa in rabbits caused an antifertility effect (fertilization failure or possibly early embryonic death), when used to treat the bull semen prior to insemination of heifers. Fertilization was prevented in rabbits inseminated with semen treated with cattle antirabbit semen serum. Normal fertility occurred in both species when the semen was treated with normal sera. Fertilization was prevented in rabbits inseminated with semen treated with the gamma-globulin fraction of the immune serum, but not with the gamma-globulin fraction of the normal serum.
Absorption of the rabbit anti-bull-sperm sera and the cattle antirabbit-semen sera with the erythrocytes of bulls and male rabbits, respectively, failed to remove the sperm agglutinins or the antifertility effect of these antisera. Absorption of these sera with the appropriate washed spermatozoa removed the agglutinins and the antifertility effect. Antisera to erythrocytes did not possess either sperm agglutinins or the antifertility effect when used to treat bull or rabbit semen. Similarly, antisera to semen or washed spermatozoa had little or no specific agglutinins against erythrocytes. These results indicate that antibodies against semen or washed spermatozoa can prevent fertilization, or may cause embryonic death. Further, they indicate the absence of cross-reactivity between the antigens of either seminal plasma or spermatozoa and erythrocytes.
ANANT P. LABHSETWAR, W. E. COLLINS, W. J. TYLER and L. E. CASIDA
The effects of oxytocin and of progesterone on the pituitaryovarian relationships were studied in sixteen non-pregnant heifers. Subcutaneous injection of 100 mg of progesterone in corn oil daily for 35 days from Day 7 of the oestrous cycle depressed both the size of the largest follicle and the total weight of follicular fluid. The fsh level was significantly raised after progesterone injections as compared to Day 1 of the cycle but not as compared to Day 7. The progesterone treatment had no significant effect on lh level. It was concluded that progesterone curtails the production, and consequently the release, of fsh and lh.
The injection of 150 U.S.P. units of oxytocin daily from the day of oestrus to Day 6 of the cycle produced a smaller corpus luteum (2·51 versus 4·40 g, P < 0·05) and a reduced concentration of progesterone (14·6 versus 29·0 μg/g, P<0·05). The oxytocin treatment had no significant effect on the hypophyseal levels of fsh and lh. It did not affect the size of the largest follicle or the total follicular fluid weight.
The corpus luteum of the previous cycle, still present on the day of oestrus, was small (1·74 g) but contained a detectable amount of progesterone (2·8 μg/g.
ANANT P. LABHSETWAR, W. E. COLLINS, W. J. TYLER and L. E. CASIDA
An experiment involving twenty multiparous Holstein cows was designed to study the pituitary-ovarian relationship in periparturient cows. The cows were divided into four equal groups. One group was slaughtered between 260 and 265 days of gestation; the second group was slaughtered within 18 hr following parturition; the third group was slaughtered 21 days post partum and the fourth group received 100 mg of progesterone daily for 20 days starting from the day of calving and was slaughtered on Day 21. There was no detectable amount of progesterone in the corpora lutea of pregnancy on the day of calving. An average of 26 μg/g progesterone was present in the corpora lutea of ante-partum cows. The corpora lutea were significantly larger in the ante-partum group than in the day-of-calving group. A significantly higher fsh level and lower lh level was found in the pituitaries of the day-of-calving group. Follicular size was significantly lower in the ante-partum and day-of-calving group as compared to both the post-partum groups. Injection of progesterone produced no significant alterations in fsh and lh levels of the pituitary glands. Neither did it depress ovarian follicular growth.
J. P. P. Tyler, P. L. Matson, W. P. Collins and M. Dukes
Summary. Treatment of mice with a dihydropyridazinone derivative (6-(4′-aminophenyl)-4,5-dihydro-5-methylpyridazin-3-one; ICI 109,081) inhibited the maturation of oocytes in the presence of gonadotrophins without affecting ovulation. The spontaneous resumption of meiosis in oocytes cultured in vitro was reversibly inhibited. ICI 109,081 may therefore be useful for evaluating the relationship between follicular development and the resumption of meiosis.