An in vitro technique to study penetration of hamster oocyte-cumulus complexes by using physiological numbers of sperm

We have developed an inexpensive in vitro system for studying cumulus penetration and fertilization by using physiological numbers of sperm. This system simulates conditions believed to exist in vivo more closely than any in current usage. In this system, 1–100 hamster sperm are used to challenge fresh hamster oocyte-cumulus complexes (OCC). Only fresh (nonoviducal) OCC are used, as they present the most stringent challenge to sperm. Because sperm numbers are low, OCC do not disperse, and sperm can be studied microscopically during penetration of the cumulus oophorus and corona radiata. These conditions permit microscopic assessment of the sperm acrosome. Video tapes of experiments allow easy review and analysis of experiments. Results obtained employing this technique show that, in vitro, (1) capacitated, acrosome-intact hamster sperm can penetrate the extracellular matrix between cumulus cells and bind to the zona pellucida; (2) the “figure-eight motility” characteristic of hyperactivated hamster sperm swimming in culture medium is suppressed when sperm swim in the extracellular matrix between cumulus cells; and (3) fertilization occurs in capillary tubes when low numbers of sperm are used. The in vitro system that we have described will be useful in analyzing the mechanisms used by sperm to penetrate the cumulus and corona radiata and to clarify the role of the acrosomal enzymes in fertilization.     

Ethanol inhibits human and hamster sperm penetration of eggs

The effect of alcohol on the fertilizing ability of both human and hamster spermatozoa was examined by an in vitro fertilization assay using hamster ova. Spermatozoa were incubated in capacitating media for 3 hr (hamster sperm) and 4 hr (human sperm). Hamster ova were inseminated with preincubated sperm and were examined after 2 to 3 hr. Ethanol was added to the capacitating media at concentrations of 25, 50, 100, 200, and 400 mg%. Fertilization of zona-free hamster eggs by human spermatozoa was reduced from 49.6% in no alcohol to 16.7% in 400 mg% ethanol. Fertilization of hamster eggs by hamster sperm revealed a reduction from 63.6% to 33.7% in cumulus-intact eggs and from 65.8% to 10.8% in cumulus-free eggs in the presence of ethanol at 400 mg%. Hamster sperm acrosome reaction was reduced from 47% to 12%. When these hamster sperm with reduced acrosome reaction were placed with zona-free hamster eggs, the 100% fertilization rate was not reduced; however, the fertilization index, which reflects the number of swelling sperm heads per egg, was reduced from 8.5 to 1.8. This suggests that as little as 12% of the sperm with an acrosome reaction is sufficient to fertilize 100% of the zona-free eggs. If ethanol was added to the insemination media only, there was no inhibition of fertilization by human sperm or hamster sperm that had been previously capacitated in an ethanol-free media. Removal of the ethanol from the preincubated sperm produced fertilization at control levels; thus the inhibitory effect is reversible. These results indicate that ethanol may affect fertilization by an inhibition of the capacitation and/or acrosome reaction process.     

Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa

Caudal epididymal spermatozoa of golden hamsters were incubated in capacitation medium. Their movement patterns changed as they became hyperactivated and underwent the acrosome reaction. To understand the basic mechanism by which changes in movement pattern are brought about, digital image analysis was carried out on the flagellar movements recorded with a video system. The degree of flagellar bending increased with incubation time, especially in the proximal midpiece. The hyperactivated spermatozoa had remarkably asymmetrical flagellar waves of large amplitude because either the bends in the same direction as the hook of the head (referred as the "pro-hook bend") or the bends in the opposite direction to the hook of the head (referred as the "anti-hook bend") extremely increased their curvature; whereas, the acrosome-reacted spermatozoa had relatively symmetrical flagellar waves of large amplitude because both the pro- and anti-hook bends remarkably increased their curvature. Beat frequency significantly decreased while wavelength of flagellar waves increased after hyperactivation and further after the acrosome reaction. These results suggest that both extreme pro- and anti-hook bends are essential in the acrosome-reacted spermatozoa even though beat frequency decreased markedly.     

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2+

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn²⁺ in millimolar concentrations, whereas in the female reproductive tract, the concentration is around 1 µM. In this study, we characterize the role of Zn²⁺ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of GPR39‐type Zn‐receptor localized mainly in the sperm tail. Zn²⁺ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39–adenylyl cyclase (AC)–cyclic AMP (cAMP)–protein kinase A–tyrosine kinase Src (Src)–epidermal growth factor receptor and phospholipase C. Both the transmembrane AC and the soluble‐AC are involved in the stimulation of HAM by Zn²⁺. The development of HAM is precisely regulated by cAMP, in which relatively low concentration (5–10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn²⁺ were added to the cells; low Zn²⁺ stimulated HAM, whereas at relatively high Zn²⁺, no effect was seen. We further demonstrate that the Ca²⁺‐channel CatSper involved in Zn²⁺‐stimulated HAM. These data support a role for extracellular Zn²⁺ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.     

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2+

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn²⁺ in millimolar concentrations, whereas in the female reproductive tract the concentration is around 1 µM. In this study, we characterize the role of Zn ²⁺ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of G protein‐coupled receptor 39 (GPR39) type Zn‐receptor localized mainly in the sperm tail. Zn ²⁺ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39‐AC‐cAMP‐PKA‐Src‐EGFR and phospholipase C. Both the transmembrane adenylyl cyclase (AC) and the soluble‐AC are involved in the stimulation of HAM by Zn ²⁺. The development of HAM is precisely regulated by cyclic adenosine monophosphate, in which relatively low concentration (5–10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn ²⁺ were added to the cells; low Zn ²⁺ stimulated HAM, whereas at relatively high Zn ²⁺, no effect was seen. We further demonstrate that the Ca ²⁺‐channel CatSper involved in Zn ²⁺‐stimulated HAM. These data support a role for extracellular Zn ²⁺ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.     

Hamster sperm motility transformation during development of hyperactivation in vitro and epididymal maturation

The transformation of hamster sperm motility during capacitation in vitro and during maturation in the caudal epididymis was analyzed and compared using videomicrography. Sperm recovered from the distal portion of the caudal epididymis, as well as ejaculated sperm recovered from the uterus exhibited low amplitude, planar flagellar beating. By 3 hr of incubation under capacitating conditions, the caudal epididymal sperm were swimming in helical patterns apparently produced by significantly increased acuteness of flagellar bending and by torsion seen as abrupt, periodic turning of the head. By 4 hr, most sperm were hyperactivated, swimming in circles resulting from asymmetrical, planar flagellar bending that was significantly more acute than the preceding patterns. When motility parameters of fresh sperm were compared with those of sperm swimming in the transitional helical pattern and with hyperactivated sperm, transitional sperm had significantly higher net and average path velocities than the others, indicating that they covered space at the greatest rate. This suggests that the transitional phase plays an important role in sperm transport. Sperm recovered from the proximal region of the caudal epididymis, near the corpus, swam in either the helical or hyperactivated patterns, or a mixture of the two. The means of their flagellar curvature ratios and linear indices were intermediate between helical and hyperactivated mean values. Thus, sperm undergoing final maturation in the caudal epididymis reverse the pattern of development of hyperactivation. Also, the development of hyperactivated motility must therefore entail induction of a preexisting potential for flagellar movement, rather than a maturational process.     

Role of the major ecto-phosphoprotein in sperm flagellar motility using a cell electroporation method

Previous studies from our laboratory have identified MPS, a 100-kDa protein, as the major phosphoprotein substrate of caprine sperm ecto-cyclic AMP independent protein kinase. In this study the isolated (32)P-labelled MPS has been incorporated into mature caprine (Capra indicus) cauda-epididymal spermatozoa with the help of cell electroporation technique to investigate the effect of MPS on sperm flagellar motility. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2 x 10(8)/ml) to external electric field of intensity 1.5 kV/cm and capacitation of 25 microF at 4 degrees C and post-pulse incubation at 37 degrees C for 1 hr. when nearly 50% of the cells lost motility. Scanning electron micrographs (SEM) demonstrate the formation of micro-pores and local osmotic swelling in the electroporated spermatozoa. MPS incorporation was maximal when its concentration was 30 microg/ml (300 pmol) in the medium and when the post-pulse incubation time was 60 min. At maximum (75%) MPS incorporation, total and forward motility increments were also maximum: 34% (P < 0.01) and 32% (P < 0.01), respectively. The subcellular fractionation data show that major portion of the introduced MPS was bound to the plasma-membrane of spermatozoa. The 32P-labelled electrophoresed intact spermatozoa lost radioactivity due to the action of the endogenous ecto-phosphoprotein phosphatase. Therefore MPS is primarily localised on the sperm external surface leaving its phosphate group(s) oriented in the extracellular medium. The data provided further evidence to strengthen the view that MPS is an ecto-phosphoprotein and that it plays an important role in the regulation of sperm flagellar motility.     

Rhesus monkey sperm penetration into zona-free hamster ova: Comparison of preparation and culture conditions

A major problem in development of nonhuman primate in vitro fertilization is the selection of donor males and repeated collection of consistent sperm samples. In practice, collection of a viable semen sample is highly dependent on operator technique and the type of animal restraint. We report an updated method for semen collection from the rhesus monkey (Macaca mulatta), use of TES-Tris (TEST) Yolk Buffer (TYB) for prolonged sperm storage and improved results of hamster ovum penetration assay. Semen was obtained from adult males restrained with 2.0 mg/kg IM ketamine hydrochloride prior to direct penile stimulation (Grass SD-9, frequency 150, delay 9, duration 7, volts 12–18, repeat mode, twin pulse). Liquified semen was washed and centrifuged twice at 100 × g for 5 min in BWW, Ham's F-10 and TALP and allowed to swim-up 60 min at 37° in 5% CO₂ and air. Alternatively, semen was mixed 1:1 with TYB, refrigerated 20 h at 4°C, centrifuged at 100 × g for 5 min, and the pellet resuspended in 1.0 ml of TALP or BWW prior to use. Hamster ova penetration was achieved with capacitated macaque sperm. Penetration was significantly improved (P < .001) with preincubation in TYB followed by resuspension in TALP (79%).     

Characterization of a spectrophotometric technique for the estimation of fowl and turkey sperm motility

An objective method of estimating the motility of fowl and turkey spermatozoa, depending on their rheotactic and light-scattering properties, has been developed. From a 1- to 2-minute recorder trace of the optical density of diluted semen before and after stopping its passage through a flow cuvette, three independent constants may be simply and graphically determined. These are: OD_m, the maximum optical density of semen flowing through the cuvette, shown to be dependent on the concentration of spermatozoa; % (ΔOD)_m, the maximal change of optical density following cessation of flow, which has been correlated with the percentage of motile spermatozoa in the sample; and t½, the time taken for the change of optical density to reach % (ΔOD)_m. This latter parameter has been correlated with forward motility of both fowl and turkey spermatozoa.     

Regional specialization of the sperm membrane in the tick Amblyomma hebraeum koch (Acari: Ixodidae)

Summary The spermatozoon of Amblyomma hebraeum is about 200 m long and comprises: (1) a thick, club-shaped anterior part, about 20 m long bearing at its apex a tactile hemisphere, and (2) an elongated tail-like part, about 180 m long. The surface of the tactile hemisphere is covered by numerous bulbous expansions, attached to it by short stalks. The base of the hemisphere is surrounded by a fringe of thin motile processes; the remaining surface of the spermatozoon is covered with long cellular processes which run more or less parallel to one another.The membrane-associated particles found on the membrane beneath the cellular processes are regularly arranged as groups of parallel strands. The external surface of the so-called peripheral granules, as revealed by freeze-etching, is smooth with a very small number of particles. Internally the particles exhibit a regular hexagonal pattern which has not been observed, so far, on any other membrane of these sperm cells.The regional specialization of the spermatozoon surface membrane in relation to sperm motility is discussed. The results obtained indicate that processes of three types: (1) bulbous expansions, (2) motile processes, and (3) cellular processes are regional specializations, all engaged in aspects of sperm motility.The technical assistance of Mr. R. Haemmerle of Balzers Research Laboratories, Mrs. F. Seif and Miss C. Pugin, is gratefully acknowledged     

达氏鲟精子的主要生物学特性

达氏鲟(Acipenser dabryanus)属淡水定居性鲟鱼类,为我国特有种,主要分布在长江上游干流及金沙江下游。长期人为过度捕捞及其生存环境的持续污染和水利工程的影响,使得达氏鲟自然种群资源遭到严重破坏,其配子质量的下降己经成为限制其规模化人工繁殖成功的关键因素之一,因此为解决达氏鲟规模化人工繁殖过程中存在的关键性技术点,作者从达氏鲟精液基本特征、精浆元素组成以及不同水体和Na+、K+对达氏鲟精子活力的影响、精子超微结构方面入手,对达氏鲟精子的生理生态特性进行了研究。结果显示,达氏鲟精子平均密度为1.52×109个/ml;精浆元素以Na+含量最高,其次是K+,之后为Ca2+、Mg2+、Cu2+、Zn2+,其中Na+、K+、Zn2+在达氏鲟精浆中的含量有极显著性差异(P0.01),Ca2+、Cu2+、Mg2+差异不明显;精子在江水中的活力最高;在Na+浓度为20 mmol/L时,精子活力最高,精子快速运动时间(FT)和寿命(LT)分别为(66.7±7.1)s和(177.0±14.9)s;达氏鲟精子对K+浓度变化较为敏感,在K+浓度为0.05 mmol/L时,精子FT和LT最长,分别为(109.0±16.1)s和(189.3±12.4)s,K+浓度超过0.05 mmol/L后精子FT和LT急速下降,当K+浓度达到0.5 mmol/L以上时,精子活力立即受到抑制;达氏鲟精子细胞核长(5.67±0.20)μm,鞭毛长(63.16±2.79)μm,全长为(70.35±2.92)μm。     

The evolution of hamster sperm motility during capacitation and interaction with the ovum vestments in vitro

Movement characteristics of golden hamster spermatozoa were studied upon collection from the cauda epididymis, during an incubation which capacitates the spermatozoa in vitro, during penetration of the cumulus, and during attachment to and penetration of the zona pellucida. High-speed videomicrography was employed to quantitate flagellar beat frequency and shape. The status of the acrosome was also assessed. During capacitation, hamster spermatozoa become increasingly invigorated before the onset of hyperactivated motility. Within the cumulus, beat frequency and curvature are reduced, apparently in response to the physical resistive properties of the matrix material. These properties appear to vary within the cumulus. Initial attachment to the zona precedes completion of the acrosome reaction, is non-rigid, and is accompanied by increased beat frequency and curvature. Subsequently, the onset of rigid binding to the zona, completion of the acrosome reaction, and increased flagellar beat frequency are very closely associated in time. The latter produces an increase in thrust against the zona. Preliminary results indicate that ensuing zona penetration requires not more than five minutes, is at oblique angles, and is associated with a continuation of vigorous flagellar beating.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: I. A fertility assay for fresh semen

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona-free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within-liposome-concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of ?.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was ?.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona-free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.     

Changes in the rat sperm head during epididymal transit

Morphological changes in sperm are one aspect of a maturation process during epididymal transit in marnrnals. The literature mentions only, for different strains of rats, a remodeling and decrease in size of the acrosome. In the present work, the sperm were obtained from caput, corpus, and cauda epididymis of the albino rat. Samples were processed for scanning electron microscopy, with routine techniques, and for light microscopy and video microscopy. It appeared, with these techniques, that the acrosomal curvature and the whole head surface area of the rat sperm decrease during the epididymal transit. To measure these changes, a geometrical method was designed, and surface measurements were made using a computer program. It was found that the caput sperm head has the greatest surface area and a sharper acrosome bend than the cauda sperm. In an attempt to explain the above-mentioned changes, the suggestion is offered that some compactation of the nucleus and acrosomal material could be related to the decrease of the surface area.     

The three-dimensional nature of the sperm tail wave studied using heterophil head-to-head agglutinins

Head-to-head agglutinates of bull spermatozoa, produced by heterophil agglutinins in human sera, revealed the same phenomenon as described for human spermatozoa: When rotating on the plate all agglutinates rotated counterclockwise. The interpretation lends further support to the hypothesis that the normal direction of the propagation of the overall helix-like tail wave is counterclockwise (ie, opposite the direction of the axonemal arms), corresponding to a clockwise shape of the wave (from base to tip) at a given instant. Heteroagglutinins may be a useful tool in physioanatomical studies of spermatozoa.     

Effects of cumulus cells on sperm penetration of bovine oocytes in protein-free medium

The present study was conducted to examine the effects of cumulus cells on sperm capacitation, acrosome reaction and penetration of bovine oocytes in vitro in a protein-free medium. In vitro matured oocyte-cumulus complexes (OCCs) and denuded oocytes were co-incubated with spermatozoa in the medium with or without bovine serum albumin (BSA). Higher fertilization rates were obtained in the OCCs (92 and 89%, respectively) than denuded oocytes (57 and 6%, respectively) in the medium with or without BSA (P<0.01). Higher proportion of the denuded oocytes were fertilized in the medium with BSA (57%) than without BSA (6%; P<0.01). These results suggest that the cumulus cells are more effective for increasing fertilization rate than BSA (P<0.05). Both the percentages of capacitated and acrosome-reacted spermatozoa incubated for 4 h with isolated cumulus cells were not significantly different in the medium without cumulus cells in the presence or absence of BSA. The denuded oocytes were inseminated with isolated cumulus cells taken from OCCs matured with or without hormones, follicle stimulating hormone (FSH) and estradiol-17beta (E(2)), and from immature OCCs in a protein-free medium. Presence of the cumulus cells matured with hormones enhanced sperm penetration of denuded oocytes more effectively (81%) than either of the cells matured without hormones (41%) or the immature cells (26%; P<0.01). The conditioned medium of cumulus cells matured with hormones was not effective for sperm penetration of denuded oocytes (2%), while a high proportion (82%) of the oocytes were fertilized when they were inseminated with isolated cumulus cells (P<0.01). In conclusion, the presence of cumulus cells matured with FSH and E(2) was effective for sperm penetration but not for sperm capacitation or acrosome reaction.     

Hyaluronidase activity of macaque sperm assessed by an in vitro cumulus penetration assay

A model system consisting of cynomolgus macaque sperm and ovulated hamster ova-cumulus complexes (OCCs) was utilized to study the role of the sperm protein PH-20 in cumulus penetration. The hyaluronidase activity of solubilized macaque sperm PH-20 was evaluated using an ELISA-like microplate assay prior to and following the addition of the hyaluronidase inhibitors heparin (0–100 μg/ml) and apigenin (250 μM), as well as the Ig fraction of a polyclonal antibody raised against purified recombinant macaque PH-20 (R10; 10–400 μg/ml). Sperm motility following exposure to enzyme inhibitors was evaluated using computer-aided sperm motility analysis. Macaque sperm were labeled with the permeant fluorescent nuclear dye, Hoechst 33342, and were coincubated with ovulated hamster OCCs for 30 min at 37°C. The addition of heparin, apigenin, or R10 antibody to solubilized sperm extracts resulted in a linear dose-dependent decrease in hyaluronidase activity (P < .01). In the heterologous cumulus penetration assay, fluorescently labeled macaque sperm that were pretreated with heparin (1–100 μg/ml), apigenin (250 μM), or R10 antibody (Ig fraction, 10–400 μg/ml) demonstrated a dose-dependent decrease in the ability to penetrate hamster OCCs (P < 0.01), in the absence of effects on sperm motility. In the homologous assay, experiments using macaque OCCs and fluorescently labeled macaque sperm confirmed that the same concentrations of heparin and R10 antibody similarly suppressed spermatozoal cumulus penetration (P < .01). These results suggest that macaque sperm PH-20-derived hyaluronidase participates in cumulus penetration in this species, and that this model system is useful for further studies into primate gamete interaction. Mol. Reprod. Dev. 46:392–400, 1997. © 1997 Wiley-Liss, Inc.     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.