In this study we evaluated the effect of several trypsin inhibitors (p-aminobenzamidine: pAB; N-α-p-tosyl-l-lysine-chloromethyl-ketone: TLCK and p-nitrophenyl-p′-guanidino-benzoate: NPGB) on sperm binding and penetration of the human zona pellucida. Motile spermatozoa, selected by a two-step Percoll gradient, were incubated at 1 × 107 cells ml−1 at 37°C and in 5% CO2 for 4.5 h. This was followed by the addition of 1 mmol pAB l−1 or phosphate-buffered saline (control) for 30 min. Three to four non-viable human oocytes were then added to each sperm suspension and incubated for 3 h. The numbers of spermatozoa bound to the human zona pellucida and in the perivitelline space were determined by phase contrast microscopy. The results showed that pAB significantly inhibited zona penetration by spermatozoa (56 ± 8% oocytes penetrated, control versus 0 ± 0% oocytes penetrated, pAB, mean ± sem), without modifying spermatozoa–zona pellucida binding. The inhibition of zona penetration was due to a block of the acrosome reaction normally induced by the human zona pellucida. In separate experiments, sperm suspensions pretreated with 1 mmol pAB l−1 or 10 μmol NPGB l−1 exhibited a marked decrease in the percentage of acrosome reactions on the zona surface (85 ± 4% and 76 ± 3% inhibition, respectively). In addition, the inhibitors prevented the acrosome reaction induced by human follicular fluid (percentage of acrosome-reacted spermatozoa: control 8 ± 2; follicular fluid 25 ± 3; pAB 6 ± 2; NPGB 8 ± 1; TLCK 12 ± 2). Electron microscope studies suggested a significant inhibition of the membrane fusion events of the acrosome reaction in the inhibitor-treated spermatozoa. These results are the first to show that trypsin inhibitors block sperm penetration of the human zona pellucida owing to an inhibition of the acrosome reaction. In addition, they suggest a role for a trypsin-like enzyme during the acrosome reaction of human spermatozoa.
M. Llanos, P. Vigil, A. M. Salgado and P. Morales
P. Morales, J. W. Overstreet and D. F. Katz
Summary. Spermatozoa from 10 fertile donors and from 10 patients with infertile marriages were washed and centrifuged (time zero, TO), and incubated in vitro in capacitation media for 6 h (T6), or 24 h (T24). At each time individual spermatozoa were classified as being morphologically normal or abnormal, and their movement characteristics were determined using high-speed videomicrography. Zona-free hamster oocytes were added to the T24 sperm suspensions. At all times, morphologically normal spermatozoa from donors and patients swam faster and had greater rolling frequency, flagellar beat frequency and amplitude than did abnormally shaped cells. Morphologically normal spermatozoa from donors exhibited a significant change in their movement pattern at T6. This change, which resembles hyperactivation in other species, was characterized by higher values of amplitude of lateral head displacement, and lower values of linearity, beat frequency and flagellar curvature ratio. In contrast, normal spermatozoa from patients showed only a decrease in straight line velocity at T6, with no other significant changes in movement characteristics. No changes in sperm movement could be demonstrated for the abnormal cells in either group of subjects. In sperm suspensions from donors and patients examined at T24, sperm vigour declined regardless of the morphological type. Spermatozoa from all 10 donors were able to penetrate the zona-free hamster oocytes, but spermatozoa from 5 of the 10 patients failed to penetrate oocytes. Correlations between hamster oocyte penetration and indicators of sperm vigour were demonstrated only for spermatozoa of patients.
Keywords: capacitation; sperm motility; hyperactivation; male fertility; sperm penetration assay
V. Alemán, R. Trejo, E. Morales, P. Hernández-Jáuregui and G. Delhumeau-Ongay
Summary. A technique was developed to obtain enriched populations of large numbers of primary spermatacytes (70–81%) and of spermatids (75%) from immature rat testes (21–23 and 38 days old respectively) in a simple and rapid fashion. The cells were nearly all viable and membrane preservation was good. The testicular cells were dispersed by a mild mechanical treatment combined with an incubation with purified collagenase, and the cell populations were separated by centrifugation in a discontinuous dextran gradient in a cell culture medium.
G Hernandez, JG Hernandez-Jimenez, P Guelmes, JE Sanchez-Criado, C Bellido, Martinez-Morales JR, L Prieto, F Marin, C Glidewell-Kenney, FJ Lopez and R Alonso
The effects of LY117018-HCl (LY; a benzothiophene similar to raloxifene) were examined on various reproductive parameters in female rats. Four-day cyclic rats were treated (10:00 h on dioestrus) with LY (0.01, 0.1, 0.5, 1, 2, 4 or 16 mg kg(-1) p.o.) and assessed for ovulation at oestrus. LY inhibited ovulation at doses as low as 0.5 mg kg(-1), and ovulation did not occur at doses of 4 and 16 mg kg(-1). LY (16 mg kg(-1)) reduced wet uterine mass and LH concentrations at the time of the expected ovulatory surge. Ovulation induced by hCG in pentobarbital-treated rats was not altered by LY treatment, indicating normal ovarian sensitivity to gonadotrophins. LY, however, completely blocked the effects of oestradiol (under either negative or positive feedback modes) on LH secretion in ovariectomized rats. GnRH secretion into hypophyseal portal blood during pro-oestrus was not affected by treatment with LY, whereas the concentrations of serum LH remained reduced. Finally, treatment with LY markedly reduced pituitary sensitivity to GnRH during pro-oestrus, as it completely blocked GnRH-induced LH secretion. These results demonstrate that LY inhibits oestradiol action in the uterus and prevents ovulation in normal cyclic rats. LY-induced inhibition of ovulation is not caused by an alteration of the ovarian response to gonadotrophins or an impairment of GnRH secretion at the hypothalamus, but by a reduction in the sensitivity of gonadotrophs to the stimulatory effects of GnRH during pro-oestrus.
I K M Liu, J W Turner Jr, E M G Van Leeuwen, D R Flanagan, J L Hedrick, K Murata, V M Lane and M P Morales-Levy
In this study of equids, we investigated the antibody response and the effect on the estrous cycle following a single inoculation of porcine zonae pellucidae (pZP) employing controlled-release methodology. We also investigated the use of two different water-soluble adjuvants as an alternative to oil-based adjuvants. Twenty-seven domestic mares were inoculated with various formulations of pZP and adjuvant. We showed that the anti-pZP antibodies generated as a result of the inoculations persisted for at least 43 weeks (length of the study). Of the various formulations used in the study, pZP and QS-21 water-soluble adjuvant, administered in combination with an emulsified preparation of pZP and Freund’s Complete Adjuvant generated a significantly (P < 0.05) higher titer of anti-pZP antibodies when compared with other formulations employing the water-soluble adjuvant, Carbopol. Hormone analyses for cyclicity indicated a high incidence and extended duration of persistent corpora lutea among the treated mares. The positive control group of mares receiving two standard inoculations of pZP and Freund’s Complete and Incomplete Adjuvants, as well as the placebo group of mares injected with QS-21 only, also exhibited high incidences of persistent corpora lutea. However, all mares eventually returned to normal cyclicity. The basis for the high incidence and extended duration of persistent corpora lutea was unexplained. The results demonstrate for the first time the persistent generation of anti-pZP antibodies following a single inoculation of pZP incorporated into a controlled-released preparation in the horse. This study further suggests that a single inoculation of pZP sequestered in a controlled-release lactide-glycolide polymer may serve as an alternative to traditional two-inoculation protocols for contraception investigations in the equine.