Hamster Egg Penetration Test Research Articles

The effect of A23187 concentration and exposure time on the outcome of the hamster egg penetration test

We investigated the conditions required to enhance the performance of human sperm in the hamster egg penetration test with the free acid form of A23187. The best performance was observed after stimulation with 2 microM A23187 for 1 h when the median penetration rate with sperm from fertile donors was 100% of eggs with 5.8 decondensed sperm heads/egg. Extending the stimulation period with 2 microM A23187 to 2 or 3 h, resulted in a progressive decrease in the penetration rate. In the absence of A23187, the penetration rate was lower (0.7 decondensed sperm heads/egg after 1 h) but increased with stimulation time. A similar picture was observed with sperm from patients taken for an IVF programme. For a pool of cryopreserved semen, the coefficient of variation of the penetration rate after stimulation with 2 microM A23187 for 1 h, expressed as decondensed sperm heads/egg, was 11% within and 20% between assays. There was no correlation between the outcome of the hamster egg penetration test and the percentage motility, velocity or lateral head displacement of the sperm measured after the same stimulation regime. However, in IVF patients the initial velocity and lateral head displacement of the sperm (zero time) were correlated with the best result from the hamster egg penetration test (r = 0.62 and 0.57 respectively). No motility changes characteristic of capacitation were detected. We conclude that stimulation with 2 microM A23187 (free acid) for 1 h prior to the addition of the zona free hamster eggs can produce a high penetration rate with fertile samples and provides a convenient and robust protocol for the assay. However, when carried out in this way the test does not assess the ability of the sperm to capacitate.

A procedure for cryopreservation of hamster oocytes yielding highly conserved oocytes suitable for sperm penetration tests

Hamsters were superovulated and their oocytes frozen in Dulbecco's phosphate-buffered saline containing 10% fetal calf serum and 1.5 molar 1,2-propanediol. In several experiments, 94% of the hamster oocytes survived freezing and were equivalent to fresh oocytes in sperm penetration. In routine use of the procedure described, also 94% of 1,340 frozen-thawed oocytes were satisfactory for sperm penetration of several species. Frozen oocytes eliminate problems and costs of maintaining hamster colonies with variable responses on individual days.

Relationship Between Hypoosmotic Swelling Test, Semen Analysis, and Zona-Free Hamster Ovum Test

The hypoosmotic swelling test is a simple test for measuring the functional competence of the human sperm membrane. Fifty-four patients with idiopathic infertility were assessed by hypoosmotic swelling test and the results were compared with those of routine semen analysis and zona-free hamster ovum human sperm penetration test (ZSPT). Semen samples with abnormal semen parameters had lower percentage of swollen sperm in comparison with those with normal semen parameters. A positive correlation was observed between sperm concentration and sperm swelling (r = .50, p less than .05). A strong positive correlation was observed between the percentage of sperm motility and the percentage of swollen sperm (r = 0.60, p less than .01), and between motile sperm concentration and sperm swelling (r = .62, p less than .01). On the other hand, sperm swelling correlated only weakly with the percentage of sperm penetration. The results indicate that the hypoosmotic swelling test appears to evaluate different functional qualities of sperm than ZSPT.

The influence of human follicular fluid on the acrosome reaction, fertilizing capacity and proteinase activity of human spermatozoa

The present study was designed to assess physiological and enzymatic changes in human spermatozoa following incubation in human follicular fluid (HFF). Initially, it was determined that infertility patients (n = 29) scored dramatically higher on the hamster egg penetration test (HEPT) when spermatozoa were incubated with HFF (22.9 +/- 4.4%) rather than the standard swim-up alone (4.6 +/- 1.1%). To further evaluate this effect, in experiment I, spermatozoa were obtained from proven fertile individuals and evaluated following exposure to three different HFF samples as well as control treatments (medium, cul de sac fluid and heparin). There were no significant differences in HEPT scores following sperm incubation in the three different follicular fluids although incubation in all the fluids significantly (P less than 0.01) enhanced sperm penetration (%PEN) when compared to the standard swim-up and other control treatments. The absence of an effect of cul de sac fluid on spermatozoa indicates that not all body fluids contain factors which stimulate sperm fertilizing capacity. The effect of HFF was demonstrated in a infertile patient population as well as in donors of proven fertility. In experiment II, the effect of HFF on the acrosome reaction (%AR), sperm fertilizing capacity and changes in sperm proteolytic enzymes were determined. There was no significant difference in the %AR between freshly ejaculated (7.9 +/- 3.1) and medium incubated (9.4 +/- 1.6) spermatozoa; however, in both of these treatments the %AR was less (P less than 0.01) than for spermatozoa treated with HFF (45.6 +/- 4.7). The %PEN following incubation of spermatozoa in HFF (52.2 +/- 6.8) was greatly increased (P +/- 0.01) compared to the standard swim-up (19 +/- 3.9).

Improvement of cervical mucus viscoelasticity and sperm penetration with sodium bicarbonate douching

The aim of the study was to evaluate the influence of vaginal douching with sodium bicarbonate (NaHCO3) upon cervical mucus viscoelasticity and sperm penetration in vitro and in vivo. Twenty-five couples with primary infertility for greater than 12 months participated in the study. The selection criteria were: (i) semen quality compatible with conception, (ii) regular ovulatory cycles and (iii) repeated negative post-coital test (PCT). After at least one inventory cycle, three consecutive cycles were studied. In the second and third cycles, vaginal douching was performed with either 1.5% (w/v) NaHCO3 or 0.9% (w/v) NaCl (randomized procedure). The viscoelasticity of the cervical mucus, sperm penetration tests (SPT) and PCTs were analysed. The viscoelasticity of mucus samples after NaHCO3 douching was significantly lower than the viscosity after NaCl douching (P less than 0.001, n = 16) and in the control cycles (P = 0.003). The SPT scores were significantly higher in the NaHCO3 cycles than in the NaCl cycles (P = 0.004, n = 22) and in the control cycles (P less than 0.001). The PCT scores proved to be significantly higher after NaHCO3 douching than after NaCl douching (P = 0.002, n = 21). Comparison of NaHCO3 and control cycles also showed a significant improvement of the PCT score after NaHCO3 douching (P less than 0.001).

Treatment of spermagglutination with proteolytic enzymes, II. Sperm function after enzymatic disagglutination

Antibody-mediated spermagglutination is responsible for infertility in some couples and fertilization in vitro can also be impaired by these antibodies. Having previously demonstrated the possibility of enzymatic disagglutination in such situations, the functional potential of disagglutinated spermatozoa has now been assessed. Chymotrypsin (500 U/ml) and papain (50 U/ml) resulted in impairment of oocyte penetration in the zona-free hamster egg penetration test. Trypsin (500 U/ml), while having no effect on egg penetration of normal spermatozoa, significantly improved oocyte penetration of spermatozoa which had been previously incubated with spermagglutinating antibody-positive sera. Sperm-mucus interaction was not improved, however, by trypsin treatment of agglutinated spermatozoa. This technique may be of value in conjunction with in-vitro fertilization in situations where spermagglutination exists, and also possibly with intra-uterine insemination if improved fertilizing ability can be confirmed in vitro.

Variation in the cholesterol/phospholipid ratio in human spermatozoa and its relationship with capacitation

The cholesterol/phospholipid (C/PL) ratio was determined for spermatozoa from eight men with normal semen parameters. There was a close correlation between the C/PL ratio and the rate of sperm capacitation as assessed by the hamster egg penetration test. A lower C/PL ratio correlated with a faster capacitation time. This supports the notion that the loss or reduction of membrane cholesterol constitutes an important step of capacitation in human spermatozoa.

The effects of lymphokines and monokines on human sperm fertilizing ability in the zona-free hamster egg penetration test

Antisperm cell-mediated immunity has been associated with infertility in men and women. The purpose of this study was to determine whether the products of activated lymphocytes and macrophages (Iymphokines and monokines) affect the fertilizing ability of human spermatozoa in the zona-free hamster egg penetration test. Of a panel of six different purified cytokines tested, the Iymphokine 'Y-interieron and the mono kine tumor necrosis factor significantly affected the ability of human sperm to penetrate hamster eggs. An inhibitory effect was observed when motile sperm were harvested for use in the assay by a swim-up technique after overnight incubation in low amounts of these cytokines and also when the factors were added to the cultures at the time sperm were introduced to the eggs. These data contribute further evidence that soluble factors produced by activated lymphocytes and macrophages in reproductive tissues could be significant mediators of immunologic infertility. (AM J OSSTET GVNECOL 1989;160:1154-9.)

The effects of lymphokines and monokines on human sperm fertilizing ability in the zona-free hamster egg penetration test

Antisperm cell-mediated immunity has been associated with infertility in men and women. The purpose of this study was to determine whether the products of activated lymphocytes and macrophages (lymphokines and monokines) affect the fertilizing ability of human spermatozoa in the zona-free hamster egg penetration test. Of a panel of six different purified cytokines tested, the lymphokine -y-interferon and the monokine tumor necrosis factor significantly affected the ability of human sperm to penetrate hamster eggs. An inhibitory effect was observed when motile sperm were harvested for use in the assay by a swim-up technique after overnight incubation in low amounts of these cytokines and also when the factors were added to the cultures at the time sperm were introduced to the eggs. These data contribute further evidence that soluble factors produced by activated lymphocytes and macrophages in reproductive tissues could be significant mediators of immunologic infertility. (Am J Obstet Gynecol 1989;160:1154-9.)

Absence of BoLA class I antigens on bovine spermatozoa.

Forty AI bulls were tested for BoLA class I antigens by means of eight specific polyclonal reagents. By means of immobilization and sperm penetration tests these antigens were not detected on sperm cells. Isoimmunization studies with the use of sperm as antigenic stimuli and insemination of frozen spermatozoa diluted in specific reagents did not prove the presence of BoLA class I antigens on bovine spermatozoa. The cytotoxic tests used in this investigation were not reliable.

Prediction of fertility of bovine semen: Preliminary studies with the hamster egg penetration test

The ability of liposome-treated fresh and frozen spermatozoa from two bulls to interact with zona-free hamster oocytes was examined to show whether the in vitro test results would correspond with in vivo fertility as indicated by the 60 to 90 d nonreturn to service rates which, using frozen semen, were 77 and 59%, respectively. The motility of spermatozoa in washed suspensions was also rated. Hamster test results were obtained using three ejaculates from each bull both as fresh and frozen semen. The results with frozen semen corresponded with fertility. The averages of three hamster tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte comparing spermatozoa from the bull with the higher fertility with spermatozoa from the bull with the lower fertility were 91% and 2.7 versus 56% and 1.4, respectively. Spermatozoa washed from frozen semen from the bull with the higher fertility interacted with hamster oocytes at the higher rate even when sperm motility was rated the same for both bulls. By contrast, fresh spermatozoa from the lower fertility bull interacted with hamster oocytes at a higher rate than spermatozoa from the higher fertility bull in six tests, comparing six ejaculates of fresh semen from both bulls. Comparing the higher fertility bull with the lower fertility bull, the average of six tests for oocyte penetration rates and mean number of spermatozoa per penetrated oocyte were 60% and 1.6 versus 89% and 3.0, respectively. This suggests that this hamster test cannot be used with fresh semen to predict relative levels of fertility of frozen semen. Also, the subjective rating of sperm motility did not correspond with the in vitro oocyte penetrating ability of the spermatozoa.

Interpretation and misinterpretation of semen parameters.

Semen analysis, which is traditionally used to evaluate male fertility, may be misleading if sample is lost or inadequately mixed. A short or excessive abstinence period may cause a low count or a high count with low motility. Damage from low temperature or delay in evaluation may decrease the reported motility. Computer-assisted semen analysis has eliminated some of the variability and has allowed the evaluation of other sperm motion variables such as sperm velocity, linearity, maximum and mean amplitude lateral head displacement, and beat-cross frequency. These measurements may also occasionally be misleading. Other tests may give a better indication of the function of sperm. The hamster egg penetration test has been used but is technically difficult, and perhaps that is why there is controversy as to the clinical value of the test in predicting subfertile sperm despite normal conventional semen analysis. Some recent data suggest that the hypo-osmotic swelling test, which determines the functional integrity of the sperm membrane, may be the appropriate prognosticator. There is a need for further studies to define the best method to determine whether a male factor is responsible for a couple's infertility.

Semen collection and analysis in the conservation of endangered nonhuman primates

In-depth investigations are needed on methods of semen collection and analysis in endangered nonhuman primates. Due to lack of resources, zoos need the assistance and support of universities and primate centers to perform these investigations. Several collection methods are described. Penile and rectal probe electrical stimulation methods for semen collection are compared. Rectal probe methods have been historically used in zoos; however, more representative samples may be acquired from primates indoctrinated to penile stimulation methods. Semen volume, concentration, pH, along with sperm morphology and motility are the commonly analyzed parameters for primates. However, with additional application to primate semen of motion analysis, viability tests, acrosomal analysis, hamster cell penetration tests, and sperm penetration tests, more accurate assessment of seminal capability may become routine. The examiner must be aware of the social and health situations of the primate under analysis, since any or all of these parameters can be adversely affected. The lion-tailed macaque (Macaca silenus) is used as an example for application of these analyses. The application of these analytical techniques to primate populations should allow better assessment about a primate's reproductive potential and the potential use of these semen samples for artificial techniques.

Acrosome reaction of human spermatozoa in zona-free hamster egg penetration test

The acrosomal status of human sperm during preparation for the process of zona-free hamster egg penetration test (ZFHEPT) was determined. The incidence of acrosome reaction (AR), as assessed by triple-stain technique, was significantly increased after 24 hours of incubation at 4 degrees C in TES-Tris (TEST)-yolk buffer, but the absolute values were relatively low (20% or less). Sperm from fertile donors and infertile patients with normal or abnormal semen analysis displayed similar capacity to undergo the AR in vitro. Although a positive correlation was found between the incidence of AR and the score of ZFHEPT, a remarkable individual variation was noted. The incidence of AR in freely swimming human sperm does not accurately reflect the fertilizing ability of the sperm.

Results of in vitro fertilization in women with antisperm antibodies in serum, cervical mucus, and follicular fluid.

This study was undertaken to investigate the presence of antisperm antibodies (ASA) in serum, cervical mucus, and follicular fluid (FF) of women undergoing in vitro fertilization and embryo transfer (IVF-ET). IgG and IgA ASA directed mostly against sperm head were found at similar concentrations in serum, cervical mucus, and FF of 2 of 34 patients. Ninety-one percent fertilization and 100% cleavage rates, respectively, were observed in one of the two patients. No fertilization occurred in the second patient. In both women, in vitro sperm penetration tests revealed hostile mucus and repeated postcoital tests were poor. It is concluded that the sperm-cervical mucus penetration test and mucus ASA measurements are useful in establishing the diagnosis of immunological infertility.

Calcium dependence of human sperm fertilizing ability

The Ca2+ dependency of human-sperm fertilizing ability was investigated using a modified Tyrode's medium either containing 2.4 mM CaCl2 (CA medium) or with the CaCl2 replaced by SrCl2 and 0.1 mM EGTA added to chelate any residual Ca2+ ions (SREG-medium). Ten washed sperm populations incubated in either medium for 0, 6, and 22 hours showed the same occurrence of acrosome reactions (by fluorescent lectin labelling and triple stain). A further 3-hour incubation after washing into fresh CA medium resulted in only a slight increase in acrosome reactions in both media. Eight sperm populations preincubated overnight in CA and SREG media were coincubated for 1 hour with previously salt-stored human zonae pellucidae also in the same media. Significantly more motile spermatozoa were bound to more of the zonae in CA medium (53.9% vs. 27.6% of zonae with 13.8 vs. 4.3 sperm/bound zona). In three hamster egg penetration test (HEPT) experiments, sperm populations preincubated overnight in either CA or SREG media were coincubated with hamster oocytes prepared in the same media. Only 2.1% of oocytes (1.0 polyspermy) were penetrated in SREG medium, cf., 30.9% of oocytes (1.3 polyspermy) in CA medium. These results demonstrate that while Sr2+ ions can substitute fully for Ca2+ in the capacitation and acrosome reaction of human spermatozoa, sperm-zona, and sperm-oolemma interaction seem to involve some more Ca2+-specific process(es). Furthermore, the increased HEPT fertilizing ability of human spermatozoa using overnight preincubation in SREG medium and CA medium for the test cannot be explained on the basis of differential kinetics of capacitation or the acrosome reaction.

Effects of relaxin on the fertilization capacity of human spermatozoa

Relaxin, a polypeptide hormone found in human seminal plasma, exerted favorable effects on the function of spermatozoa. This study was designed to investigate the effect of this hormone on the fertilization capacity of human spermatoxoa from 10 oligospermic men (group A), 11 asthenospermic men (group B), 10 normospermic infertile men (group C), and 10 men with verified fertility (group D). We compared the effects of purified porcine relaxin at different concentrations (0, 50, and 150 ng/ml). Parameters include motility, hypoosmolar swelling test, and zona-free hamster egg penetration test. In group A and group B, relaxin increased the results of the hamster egg penetration test and motility. In group C, results of the hamster egg penetration test and motility improved only at the concentration of 50 ng/ml. In group D, relaxin increased the results of the hamster egg penetration test only at a concentration of 150 ng/ml. Results of the hypoosmolar swelling test were not consistently altered in any study group. Relaxin appears to enhance the fertilization capacity and motility of human spermatozoa in oligospermic and asthenospermic subjects.

Heterologous penetration of zona-free hamster ova by ejaculated domestic cat spermatozoa

Hypertonic high-salt Biggers, Whitten, and Whittingham medium was compared with either a complex culture medium (synthetic tubal fluid) or a variant of Tyrode's medium for capacitating donor human spermatozoa to be used in the zona-free hamster egg penetration test. Five sperm preparations showed a slightly higher penetration rate in synthetic tubal fluid than in high-salt Biggers, Whitten, and Whittingham medium, five showed equivalent results, and only two had slightly lower penetration rates. However, in modified Tyrode's medium only one sample showed a lower penetration, while six showed comparable results and five showed higher rates of penetration. This last group included two samples that had demonstrated zero penetration in high-salt Biggers, Whitten, and Whittingham medium. Although no overall significant differences between media in the two series of experiments could be discerned with paired t tests, in view of the lower incidence of false negative results in modified Tyrode's medium and the hamster egg penetration test.

Microsurgical vasoepididymostomy to corpus epididymidis in treatment of inflammatory obstructive azoospermia

A unilateral microsurgical vasoepididymostomy utilizing 35 mm of epididymas was performed in a patient with postinflammatory epididymal obstruction. Success was verified with multiple semen analyses, the hamster egg penetration test, and a pregnancy. These results demonstrate that surgical bypass of inflammatory tubal obstruction in the distal epididymas can result in the return of normal epididymal function and fertility.

Review Article: Structure and Function of the Epididymis

Testicular spermatozoa are functionally immature in that they cannot fertilize ova. It was first demonstrated by Young [171, 172] that spermatozoa undergo certain changes as they migrate through the epididymis. He proposed that spermatozoa ripen during epididymal transit. It is now known that specific maturational changes occur in spermatozoa during epididymal transit which result in their developing the ability to fertilize ova. Concomitant with this functional maturity are changes in spermatozoal morphology, motility, chemistry, permeability, density and metabolism. It is apparent that in some way not understood these changes are necessary for sperm to achieve the ability to complete the fertilization process. When these mechanisms are understood, we may be able to effectively treat conditions such as necrospermia or abnormally low sperm motility. Furthermore, with the development of the hamster-egg penetration test a “new” type of male infertility has become evident in recent years; the inability of otherwise normal sperm to penetrate an ovum. It is during epididymal transit that this ability is normally acquired. Thus, any insight into how sperm attain the capacity to penetrate an ovum could lead to an effective treatment of patients whose sperm do not have this ability. In addition, the epididymis holds significant promise as the site of action for a male contraceptive. Thus, it is the purpose of this review to describe the structure and function of the mammalian epididymis with particular emphasis on the factors regulating sperm maturation.