Hyaluronic acid substantially increases the retention of motility in cryopreserved/thawed human spermatozoa

We have demonstrated previously that hyaluronic acid (HA) improves the velocity and the retention of motility in freshly ejaculated human spermatozoa. In the present work, we examined the effect of HA on cryopreserved/ thawed spermatozoa in four paradigms: (i) effect of HA on sperm motility and velocity in semen; (ii) stabilizing effect of HA after 4 h of incubation when the decline of sperm motility is already detectable; (iii) the duration of improved motility after the separation of spermatozoa from HA by Percoll gradient centrifugation; and (iv) motility of sperm cryopreserved in the presence of HA. HA improved the retention of sperm motility in thawed spermatozoa. Indeed, the motility values after 30 h were approximately 100% higher in the HA compared with the control samples. This effect of HA was also evident in the stabilization of spermatozoa with already declining motility. After removal of the HA from the incubation medium, significantly increased motility in the HA-exposed spermatozoa was still detectable for at least 4 h. Cryopreservation of spermatozoa in the presence of HA did not improve the recovery of motility. The data indicate that HA improves the retention of motility of cryopreserved/thawed spermatozoa, even after the removal of HA from the incubation medium. The utilization of HA will probably prove beneficial in assisted reproduction: in intrauterine insemination and in in-vitro fertilization (IVF), the extended sperm motility and velocity will enhance the fertilizing efficiency; in intracytoplasmic sperm injection (ICSI), the improved motility will facilitate the identification of viable spermatozoa. Because HA is a physiological component of the cumulus and of the female and male reproductive tracts, administration of HA should not cause ethical concerns.      

Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing

The purpose of this study was to examine if selecting a sperm population with improved motion characteristics before freezing reduces the deleterious effects of cryopreservation. Semen specimens from 15 normal donors were divided into two equal aliquots. The first aliquot received no treatment (control), and the second was processed by swim-up from a washed sperm preparation to select a sperm population with better motility and motion characteristics (swim-up). Both aliquots were cryopreserved by the liquid nitrogen vapour method. Percentage motility and motion characteristics were evaluated by computer-assisted semen analysis. Acrosome integrity as well as spontaneous and calcium ionophore-induced acrosome reactions before freezing and after thawing were assessed by fluorescein isothiocyanate conjugated peanut agglutinin combined with a supra vital dye (Hoechst-33258). Swim-up processing enabled selection of a sperm population with better motion characteristics, percentage motility and viability before freezing (P < 0.001), but with no difference in percentage of acrosome-intact spermatozoa (P = 0.63). After thawing, the swim-up specimens exhibited faster velocity and progression than untreated specimens (P < 0.001). They also had higher percentages of spermatozoa with intact acrosomes and spermatozoa able to undergo acrosome reaction in response to calcium ionophore (P < 0.05). Selecting a highly motile sperm population before freezing enhances overall post-thaw spermatozoa quality.     

Pentoxifylline induces hyperactivation and acrosome reaction in spermatozoa of golden hamsters: changes in motility kinematics

Pentoxifylline (PF) is used to enhance motility of spermatozoa from infertile human subjects. We have previously shown that 0.45 mM PF improved capacitation of spermatozoa and fertilization of oocytes in vitro in hamsters. The present study was carried out to assess PF- induced changes in motility kinematics of hamster spermatozoa by a computer-aided sperm analyser (CASA) and determine the timing of onset of hyperactivation (HA) and acrosome reaction (AR) in PF-treated spermatozoa. Motility kinematics were analysed by CASA for 0-8 h in the absence or presence of 0.45 mM PF in Tyrode's medium supplemented with lactate, pyruvate and polyvinyl alcohol (TLP-PVA) or in TLP-PVA with bovine serum albumin (TALP-PVA). Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Both in TALP-PVA and TLP-PVA, PF markedly increased SMI, especially the quality of motility (P < 0.02) by 2-3 h which was sustained up to 6 h. The motility kinematic data of PF-treated spermatozoa in TALP-PVA showed that average path velocity, curvilinear velocity and amplitude of lateral head displacement significantly (P < 0.05) increased as early as 2 h, with the expected decrease in straightness (STR) and linearity (LIN). Similar changes were also observed with PF-treated spermatozoa in TLP-PVA. Moreover, the percentage of hyperactivated spermatozoa in PF-treated samples was significantly (P < 0.001) higher than the untreated control at 2 h. To determine whether PF could induce AR, independent of bovine serum albumin, quantitative AR was assessed by observing the presence or absence of acrosomal cap on viable spermatozoa. PF significantly (P < 0.001) increased the percentage of AR as early as 2 h, reaching maximum at 4 h both in TALP-PVA (P < 0.05) and in TLP-PVA (P < 0.001). These results show that, in hamsters, PF induces early onset (by 2 h) of HA and AR and increases the proportion of spermatozoa undergoing physiological maturation.      

Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen

The aim of this study was to determine the relationship between calcium ionophore A23187-induced acrosome reaction (AR) and sperm fertilizing ability. Semen samples remaining after preparation for standard IVF were studied in 109 patients who had sperm concentrations > or =20 x 10(6)/ml. Ionophore-induced AR was performed on motile spermatozoa selected by centrifugation on a Percoll gradient. Semen analysis was performed using standard methods. Patients with higher (>50%, n = 76) fertilization rates had significantly higher ionophore-induced AR than patients with lower (<50%, n = 33) fertilization rates (49 +/- 14 versus 38 +/- 21%, P < 0.05). When the data from all patients were analysed by logistic regression, only the percentage sperm motility in insemination medium and ionophore-induced AR were significantly related to fertilization rates. Similar results were also obtained when the data from a subgroup of patients with poor (<15% normal) sperm morphology were analysed. However, when patients with normal sperm morphology > or =15% were analysed separately, only sperm count and the percentage of spermatozoa with progressive motility in semen were significantly related to fertilization rates. In conclusion, ionophore- induced AR was significantly related to fertilization rates in vitro mainly in patients with teratozoospermic semen. Tests for ionophore- induced AR may provide additional information about sperm fertilizing ability but may not indicate specific defects of the physiological AR.      

Tyrosine nitration in human spermatozoa: a physiological function of peroxynitrite, the reaction product of nitric oxide and superoxide

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO-) produced from the reaction of nitric oxide (NO.) with superoxide (O2(.-)). Since human spermatozoa are able to produce both NO. and O2(.-) during capacitation in vitro, we investigated whether spontaneous tyrosine nitration of proteins occurs in human spermatozoa and evaluated the physiological effects of peroxynitrite on sperm function. We report here that human spermatozoa, incubated for 8 h under conditions conducive to capacitation, display a reproducible pattern of protein tyrosine nitration. Several proteins with mol. wt of 105-14 kDa become increasingly tyrosine-nitrated after 15 min incubation and then minimal changes are observed. Treatment of capacitated spermatozoa with human follicular fluid or calcium ionophore causes an increase of the nitrotyrosine content of proteins at the mol. wt of 85 kDa. Moreover, exposure of spermatozoa to ONOO- (2.5-50 micromol/l) increases motility and primes spermatozoa to respond earlier to human follicular fluid. ONOO- also increases protein tyrosine nitration and phosphorylation in a concentration-dependent manner. Taken together, these results demonstrate that tyrosine nitration of sperm proteins occurs in capacitated human spermatozoa, and that low concentrations of ONOO- modulate sperm functions, emphasizing the concept that capacitation is part of an oxidative process.     

Outcome of in-vitro culture of fresh and frozen-thawed human testicular spermatozoa

The effect of in-vitro culture on the motility and morphology of fresh and frozen-thawed human testicular spermatozoa obtained from obstructive azoospermic patients and on the motility of testicular spermatozoa obtained from non-obstructive azoospermic patients was evaluated. The outcome of intracytoplasmic sperm injection (ICSI) with fresh and frozen-thawed human testicular spermatozoa was studied. The results showed that significant improvement of sperm morphology and motility was observed in culture of fresh (n = 17) and frozen-thawed (n = 15) testicular sperm samples obtained from patients with obstructive azoospermia. The motility of cultured testicular spermatozoa reached a peak at 72 h without the need for special media. In six of 20 samples obtained from patients with non-obstructive azoospermia, improvement of sperm motility was observed. When only non-motile testicular spermatozoa were cultured, they all remained non-motile (n = 9). In patients with obstructive azoospermia, fertilization rates of 80 and 81% were obtained using ICSI with fresh and frozen-thawed testicular spermatozoa respectively. Clinical pregnancies were observed in four out of nine patients with fresh testicular spermatozoa and two out of five patients after using frozen-thawed spermatozoa. When fresh testicular spermatozoa obtained from patients with non-obstructive azoospermia were used for ICSI, the fertilization rate was 68% and two out of seven patients achieved clinical pregnancies. In conclusion, the morphology and motility of fresh and frozen-thawed testicular spermatozoa in patients with obstructive azoospermia can be significantly improved after in-vitro culture. The outcome of in-vitro culture of testicular spermatozoa in patients with non-obstructive azoospermia is unpredictable. In-vitro culture of non-motile testicular spermatozoa is not successful so far. The outcome of ICSI with fresh and with frozen-thawed testicular spermatozoa was similar.      

Absence of glucose decreases human fertilization and sperm movement characteristics in vitro

The effect of glucose in a modified Ham's F10 medium (MM) without hypoxanthine, phosphate and transition metals on human fertilization and sperm survival in vitro was determined. Mature human oocytes from in-vitro fertilization (IVF) patients or Percoll-washed human spermatozoa were randomly allocated to one of the treatment groups: normal Ham's F10, MM, MM with 5 mM glucose (HGMM) and MM with 0.5 mM glucose (LGMM). Oocytes were inseminated in one of the four media for 12-20 h and checked for fertilization. Sperm were incubated likewise for 4 and 24 h, and sperm motility and sperm movement characteristics including average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), straightness (STR), and linearity (LIN) were determined using computer-assisted semen analysis. Fertilization rates were significantly lower in oocytes cultured in MM (23.8%) compared to LGMM (75.5%), HGMM (73.6%) or Ham's F10 (71.1%). Sperm characteristics after 4 h incubation in all four media were similar, except VAP, VSL, VCL and ALH were significantly lower in MM (no glucose) in comparison with the other three media. After 24 h VAP, VSL, VCL, ALH, LIN and percentage rapid spermatozoa were significantly higher in spermatozoa incubated in HGMM or Ham's F10 compared with MM or LGMM. Also after 24 h, the percentage of spermatozoa which were highly motile was greater in HGMM than in Ham's F10. Absence of glucose significantly lowered fertilization rates and sperm movement characteristics in vitro.      

Expression of Hsp60 and Grp78 in the human endometrium and oviduct, and their effect on sperm functions

BACKGROUND: Within the female genital tract, spermatozoa undergo a series of membranous and intracellular transformations to become competent at fertilizing the oocyte. In the bovine, previous studies have shown that two oviductal proteins, heat shock protein 60 (Hsp60) and glucose regulated protein 78 (Grp78), bind to spermatozoa and may be involved in this acquisition of fertilizing competence. METHODS: Immunohistochemical studies were performed on human endometrial and oviduct tissues to localize these two chaperones in the female genital tract. Human spermatozoa were incubated under capacitating conditions in the presence or absence of recombinant Hsp60 or Grp78. Following a 4-h incubation, the effects of these proteins were evaluated on sperm acrosomal integrity, motility, protein phosphotyrosine content and free intracellular calcium concentrations. RESULTS: Both chaperones were present in the uterus and oviduct epithelial cells and were shown to bind to human spermatozoa. Incubation with either exogenous Hsp60 or Grp78 did not affect sperm viability, motility or acrosomal integrity. Hsp60 partially prevented the increase in p81 phosphotyrosine content induced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and both chaperones significantly increased the sperm intracellular calcium concentration. Moreover, the progesterone-induced increase in intracellular calcium was higher when sperm were pre-treated with either Hsp60 or Grp78. CONCLUSIONS: Our study suggests that these two proteins may affect human sperm intracellular signalling pathways and capacitation.     

Effect of human cervical mucus on human sperm motion and hyperactivation in vitro.

The aim of our experiment was to examine the effect of exposure to human cervical mucus on quantitative sperm motility with specific reference to hyperactivated sperm motility. Human spermatozoa were allowed to penetrate cervical mucus for 20 min before swimming into Earle's balanced salt solution tissue culture medium for 25 min. The sperm motion characteristics were compared to those which had been obtained from a direct swim-up for 45 min. Spermatozoa treated with mucus were more 'active' than the control group. Multivariate statistical analysis indicated that cervical mucus promotes hyperactivated motility and that sperm sub-populations exposed to cervical mucus are very heterogeneous, as indicated by the numbers and motility characteristics of spermatozoa.     

An important role of actin polymerization in the human zona pellucida-induced acrosome reaction.

The effects of inhibitors of actin polymerization and depolymerization, cytochalasins and phalloidin, on the human zona pellucida (ZP)-induced acrosome reaction (AR) were investigated. Motile spermatozoa, selected by swim-up technique from normozoospermic men, were incubated in medium with or without the actin modulators. Oocytes (four per test) which had failed to fertilize in vitro were added and incubation continued for 2 h. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small-bore pipette and the status of the acrosomes was determined by fluorescein-labelled Pisum sativum agglutinin (PSA). Double immunofluorescent staining with PSA and an anti-actin monoclonal antibody illuminated the acrosomal region of acrosome-intact spermatozoa. In calcium ionophore-induced AR spermatozoa, actin staining was confined to the equatorial segment, post-acrosomal region and tail. Cytochalasins B and D significantly inhibited ZP-induced AR in a dose-dependent manner (P < 0.001). Both inhibitors had no effect on the acrosome of spermatozoa in the insemination medium. Cytochalasin B or D (10-40 micromol/l) had no effect on total percentage motile spermatozoa but decreased sperm velocity and hyperactivation. Phalloidin had no effect on the ZP-induced AR or sperm motility. In conclusion, actin polymerization plays an important role in human ZP-induced AR.     

Chelation of intracellular zinc ions affects human sperm cell motility.

The effects of two different zinc chelators, diethyldithiocarbamate (DEDTC) and calcium ethylenediaminetetraacetic acid (EDTA), in full semen samples and 'swim-up' samples were investigated. DEDTC, which crosses cell membranes, and EDTA, which does not cross cell membranes, were added to semen samples in different concentrations. Sperm cell motility parameters were assessed by computer-assisted semen analysis (CASA). It was found that very small concentrations (0.01 mM) of DEDTC immobilized the sperm cells within 80 min, while EDTA had no depressing effect at the concentrations used. In full semen samples EDTA enhanced straight line velocity (VSL) at concentrations of 1.0 and 0.5 mM; this effect was not found at higher concentrations. It is suggested that intracellular mitochondrial zinc ions play a crucial role for sperm cell motility, while loosely bound or free zinc ions in the seminal plasma exert a secondary role on human sperm cell motility.     

Andrology: Effects of nitric oxide on human spermatozoa: evidence that nitric oxide decreases sperm motility and induces sperm toxicity

Endogenous nitric oxide (NO) is an important functional mediatorin several physiological systems, including the reproductivesystem. However, when generated in excessive amounts for longperiods, mainly during immunological reactions, NO is cytotoxicand cytostatic for invading microbes, as well as for the cellsgenerating it and the tissues present around it. Since infertilityassociated with urogenital tract infection in males and femalesis also accompanied by reduced sperm motility and viability,it is possible that reduced fertility in these patients is dueto NO-induced sperm toxicity. We therefore evaluated the directeffects of NO, chemically derived from S-nitroso-N-acetylpenicillamine(SNAP, 0.012–0.6 mM) and sodium nitroprusside (SNP, 0.25–2.5mM), on the motility and viability of human spermatozoa. Furthermore,we tested whether inhibition of NO synthesis prevents spermmotility and viability by incubating washed total cells presentin the semen (spermatozoa, round cells) with N-nitro-L-arginine-methyl-ester(L-NAME), a NO synthesis inhibitor. Treatment of purified spermatozoawith SNAP or SNP decreased forward progressive sperm motilityand straight line velocity, and also increased the percentageof immotile spermatozoa in a concentration-dependent manner.Furthermore, the percentage of immotile spermatozoa positivelycorrelated with the percentage of dead spermatozoa. In contrastto freshly prepared SNAP, SNAP preincubated for 48 h had noeffect on the motility and viability of the spermatozoa. Furthermore,as compared to untreated controls, a significantly higher percentageof forward progressive sperm motility as well as viability (P< 0.05) was maintained in washed semen incubated with L-NAME(0.15 mM). Seminal plasma concentrations of nitrite-nitrate(stabile metabolites of NO/10⁶ spermatozoa correlated positively(P < 0.05) with the percentage of immotile spermatozoa. Ourresults suggest that NO can cause sperm toxicity as well asinhibit sperm motility. In conclusion, excessive NO synthesisin response to infection and inflammation could be an importantfactor contributing to functional change of the spermatozoa,leading to their dysfunction and to infertility.     

Angiotensin II in human seminal fluid

The renin-angiotensin system (RAS) and angiotensin II are important in sperm function and male fertility. Angiotensin II type I (AT1) receptors have been identified in developing and ejaculated human spermatozoa, and angiotensin can stimulate sperm motility, the acrosome reaction and binding to the zona pellucida. However, there is little information on the availability of the hormone to spermatozoa during the reproductive process. Seminal plasma and blood plasma obtained from normal and subfertile subjects was extracted, and angiotensin content was analysed by radioimmunoassay. Values obtained for blood angiotensin II were within the normal range at 16.0 +/- 3.1 pg/ml (mean +/- SEM). Values for seminal plasma were usually 3-5 fold higher, at 51.6 +/- 9.3 pg/ml (n = 34, P < 0.0001). High performance liquid chromatography analysis showed that approximately 80% of the immunoreactive angiotensin was attributable to angiotensin II itself. However, seminal plasma angiotensin II concentrations were not correlated with blood angiotensin II, sperm concentration or sperm motility. The results show that immunoreactive angiotensin from a source other than the circulation is available to spermatozoa in human ejaculates. The results are consistent with the concept that angiotensin II has an important role in male fertility.     

Endeometriosis: The influence of peritoneal fluid from patients with minimal stage or treated endometriosis on sperm motility parameters using computer-assisted semen analysis

The aim of this study was to determine the influence of peritonealfluid from patients with minimal stage or treated endometriosison sperm motility parameters. Peritoneal fluid aspirated atdiagnostic laparoscopy for unexplained infertility from womenduring the luteal phase of the menstrual cycle (days 20–23)was incubated for 5 h with fresh semen samples obtained frommen of recently proven fertility. Spermatozoa were preparedby a swim-up technique from unprocessed semen. Using computer-assistedsemen analysis (Hamilton-Thorn Research, MA, USA), sperm motilityand motion parameters were observed at 0, 120, 180 and 300 min.Compared with spermatozoa incubated in Earle's balanced saltsolution/human serum albumin, the percentage motility, percentageprogressive motility and progressive velocity of spermatozoaincubated in peritoneal fluid from patients without visibleendometriosis were significantly higher (P< 0.05). Maximaleffect was observed at 3 h and maintained until 5 h. We concludethat in an in-vitro study, in contrast to peritoneal fluid frompatients with minimal stage endometriosis, peritoneal fluidfrom patients with unexplained infertility and no visible endometriosiscan improve sperm motility when compared with culture medium.     

Correlation between intracellular cAMP content, kinematic parameters and hyperactivation of human spermatozoa after incubation with pentoxifylline

Various compounds have been used in the attempt to improve sperm motility, including pentoxifylline (PF), a methylxanthine derivative. It has been postulated that PF, being a phosphodiesterase inhibitor, increases sperm kinematic parameters and the number of spermatozoa exhibiting hyperactivated motility by raising the intracellular content of cAMP, a molecule involved in the generation of sperm energy. However, it has not been clarified whether the biological effects of PF on sperm motility correlate with its ability to increase intracellular cAMP levels. To examine this relationship, the kinematic parameters, hyperactivation, and intracellular cAMP content were evaluated in motile spermatozoa, obtained by discontinuous Percoll gradient and swim- up from 21 normozoospermic semen samples, incubated without and with PF for 0, 1, 2, and 4 h. PF increased beat cross frequency after 1 and 2 h of incubation, curvilinear velocity and lateral head displacement (ALH) after 4 h, and hyperactivation after 1, 2, and 4 h, and decreased linearity (LIN) after 1 h of incubation. The intracellular cAMP content of spermatozoa incubated with PF increased at all time-points examined. Both intracellular cAMP content and increase in hyperactivation in response to PF decreased with the length of incubation. In the absence of PF, cAMP content was unchanged and was correlated significantly only with ALH and the percentage of spermatozoa with hyperactivated motility. Following incubation with PF, cAMP content correlated with hyperactivation and all sperm kinematic parameters, with the exception of LIN and straightness. These findings suggest that the beneficial effects of PF on sperm kinematic parameters and hyperactivation are related to its ability to increase intracellular cAMP content.      

The yield, motility and performance in the hamster egg test of human spermatozoa prepared from cryopreserved semen by four different methods.

Different procedures were investigated for the dilution of human cryopreserved semen and the preparation of an enriched population of motile spermatozoa for assisted reproduction. The dilution of a 0.25 ml straw of cryopreserved human semen by addition of 2.0 ml Ham's F-10 buffer in one step caused a large decrease in the proportion of motile spermatozoa. This was due to osmotic stress because many of the diluted spermatozoa exhibited swollen tails. To a large extent the damage could be avoided by adding the buffer in 0.10-ml aliquots at 30-s intervals. Spermatozoa obtained after such dilution of cryopreserved human semen were subjected to the swim-up procedure, to centrifugation on two-step gradients of Nycodenz or Percoll, or to filtration through glass fibre paper and compared with respect to yield, motility parameters and penetrating ability in the hamster egg test. The swim-up procedure yielded spermatozoa with excellent motility but only 12% of the available motile spermatozoa were recovered. On both Nycodenz and Percoll gradients, greater than 40% of the available motile spermatozoa were recovered and the average velocity of the spermatozoa was not significantly less than for the swim-up technique. When A23187 was used to promote acrosome reactions in the hamster egg test, Percoll-prepared spermatozoa achieved an average of 8.6 decondensed sperm heads/egg compared to 1.9 for Nycodenz and 1.3 for the swim-up procedure. The yield from glass fibre paper filtration was only 12% and the velocity of the spermatozoa and their performance in the hamster egg test was significantly poorer than in all the other methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules.

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+]i) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline (0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoa). cGMP values ( approximately 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/ HCO3- antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 microM). A protein kinase C inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR. A phospholipase A2 inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+/-]i was measured using fura-2-AM, there was an early rise (271 nM at 0.5 h) in pentoxifylline-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR (20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-]i and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A2 and protein kinase C activity.     

The effects of human skin fibroblast monolayers on human sperm motility and mouse zygote development.

A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified.     

A new test for the assessment of sperm- zona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro

The spermatozoa of some patients attending for in-vitro fertilization(IVF) fail to penetrate the zona pellucida in vitro. A testhas been devised to identify these cases. It is based on thenumber of spermatozoa penetrating into the zona pellucida, whichwere counted after removing spermatozoa bound to the zona surfaceby vigorous aspiration of each oocyte through a narrow gauge(120 µm) glass pipette. The oocytes were collected from197 patients undergoing IVF treatment with their own gametes;79 with no oocytes fertilized and 118 with some oocytes fertilized.Sperm motility, morphology and DNA normality (acridine orangestain) were also measured. The relationships between sperm testresults and IVF rate were examined by logistic regression. Theproportions of penetrated zonae, normal sperm morphology andnormal DNA were the most significant factors related to IVFrate in the whole group. Also, in patients with 30 spermatozoabound per zona pellucida or with normal sperm morphology 30%,the proportion of penetrated zonae and normal DNA were mostsignificant. Oocytes from 42 patients who had zero fertilizationand low sperm-zona binding (average, 2.2 spermatozoa/zona pellucida)were re-incubated with normal donor spermatozoa: large numbersof spermatozoa bound (average, 88 spermatozoa/zona pellucida)and each zona was penetrated by at least one spermatozoon. Inconclusion, the percentage of zonae penetrated was the variablemost significantly correlated with IVF rate. Penetration ofthe zona was also strongly related to fertilization rates inpatients without defects of sperm morphology and sperm-zonabinding. In patients where all zonae were penetrated, poor fertilizationmay be due to sperm morphology and DNA abnormalities. Failureof sperm-zona binding and penetration in vitro in patients withfailure of fertilization was mainly due to sperm defects andnot oocyte defects     

Caprine cauda epididymal sperm motility pattern in the capacitation medium supplemented with leptin

Leptin is an adipocyte-derived hormone with a role in the reproductive system through metabolic signals. Studies have suggested that binding of leptin to its receptors which have been found in human and boar sperm activates biochemical pathways leading to sperm capacitation. The present study was designed to explore the effects of leptin on sperm kinetic pattern during capacitation in vitro. Epididymal spermatozoa were obtained from 30 adult goat testes in six replicates and incubated in capacitation medium (Ca²⁺-free Tyrode’s medium, Sperm-TALP) supplemented with 0 (Con, control group), 10 and 100 ng/mL (L10 and L100 groups, respectively). Motion parameters were assessed using computer-assisted sperm analysis system at 10, 30, 60, 120 and 240 min after sperm incubation at 37°C and 6 % CO₂. Exposure of spermatozoa to two different doses of leptin caused a significant increase (p?<?0.05) in total and progressive sperm motility, curvilinear and straight line velocity, linearity (Lin), mean angular displacement, amplitude of lateral head displacement (ALH) and beat cross-frequency (BCF) compared to the control group specially up to 60 min. There was not any significant difference between L10 and L100 groups in the evaluated parameters except for the first 10 min that L100 had better result in increasing Lin, ALH and BCF (p?<?0.05) and at the time of 240 min in which L10 showed a significant increase in all motion parameters. In conclusion, this study showed that leptin supplementation had significant effects on sperm motility patterns and so could enhance the sperm capacitation process.