BAG5 regulates HSPA8-mediated protein folding required for sperm head-tail coupling apparatus assembly.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.

Wampa is a dynein subunit required for axonemal assembly and male fertility in Drosophila.

In cilia and flagella, dyneins form complexes which give rise to the inner and outer axonemal arms. Defects in the dynein arms are the leading cause of primary ciliary dyskinesia (PCD), which is characterized by chronic respiratory infections, situs inversus, and sterility. While the pathological features associated with PCD are increasingly well characterized, many of the causative genetic lesions remain elusive. Using Drosophila, here we analyze genetic requirements for wampa (wam), a previously uncharacterized component of the outer dynein arm. While homozygous mutant animals are viable and display no morphological defects, loss of wam results in complete male sterility. Ultrastructural analysis further reveals that wam mutant spermatids lack the axonemal outer dynein arms, which leads to a complete loss of flagellar motility. In addition to a role in outer dynein arm formation, we also uncover other novel microtubule-associated requirements for wam during spermatogenesis, including the regulation of mitochondrial localization and the shaping of the nuclear head. Due to the conserved nature of dyneins, this study advances our understanding of the pathology of PCD and the functional role of dyneins in axoneme formation and other aspects of spermatogenesis.

Non-canonical RNA polyadenylation polymerase FAM46C is essential for fastening sperm head and flagellum in mice†.

Family with sequence similarity 46, member C (FAM46C) is a highly conserved non-canonical RNA polyadenylation polymerase that is abundantly expressed in human and mouse testes and is frequently mutated in patients with multiple myeloma. However, its physiological role remains largely unknown. In this study, we found that FAM46C is specifically localized to the manchette of spermatids in mouse testes, a transient microtubule-based structure mainly involved in nuclear shaping and intra-flagellar protein traffic. Gene knockout of FAM46C in mice resulted in male sterility, characterized by the production of headless spermatozoa in testes. Sperm heads were intermittently found in the epididymides of FAM46C knockout mice, but their fertilization ability was severely compromised based on the results of intracytoplasmic sperm injection assays. Interestingly, our RNA-sequencing analyses of FAM46C knockout testes revealed that mRNA levels of only nine genes were significantly altered compared to wild-type ones (q < 0.05). When considering alternate activities for FAM46C, in vitro assays demonstrated that FAM46C does not exhibit protein kinase or AMPylation activity against general substrates. Together, our data show that FAM46C in spermatids is a novel component in fastening the sperm head and flagellum.

SPATC1L maintains the integrity of the sperm head-tail junction.

Spermatogenesis is a tightly regulated process involving germ cell-specific and germ cell-predominant genes. Here we investigate a novel germ cell-specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9-mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z-line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L-PKA complex in maintaining the stability of the sperm head-tail junction, thereby revealing a new molecular basis for sperm head-tail integrity.

A comparative study of the morphometry of sperm head components in cattle, sheep, and pigs with a computer-assisted fluorescence method.

The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle (Bos taurus), sheep (Ovis aries), and pigs (Sus scrofa). Semen smears of twenty ejaculates from each species were fixed and labeled with a propidium iodide-Pisum sativum agglutinin (PI/PSA) combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed. The use of the PI/PSA combination and CASA-Morph fluorescence-based method allowed the capture, morphometric analysis, and differentiation of most sperm nuclei, acrosomes and whole heads, and the assessment of acrosomal integrity with a high precision in the three species studied. For the size of the head and nuclear area, the relationship between the three species may be summarized as bull > ram > boar. However, for the other morphometric parameters (length, width, and perimeter), there were differences in the relationships between species for sperm nuclei and whole sperm heads. Bull sperm acrosomes were clearly smaller than those in the other species studied and covered a smaller proportion of the sperm head. The acrosomal morphology, small in the bull, large and broad in the sheep, and large, long, and with a pronounced equatorial segment curve in the boar, was species-characteristic. It was concluded that there are clear variations in the size and shape of the sperm head components between the three species studied, the acrosome being the structure showing the most variability, allowing a clear distinction of the spermatozoa of each species.

A human morphologically normal spermatozoon may have noncondensed chromatin.

According to numerous assisted reproductive medicine practitioners, semen with normal characteristics might not require further investigation. However, on the scale of the individual spermatozoon, it is well known that normal morphology does not guarantee optimal nuclear quality. Here, for 20 patients with normal sperm characteristics and a high proportion of spermatozoa with noncondensed chromatin, we subsequently assessed chromatin condensation status (aniline blue staining) and morphology (Papanicolaou staining) of the same 3749 spermatozoa. Although the overall proportion of morphologically normal spermatozoa was not correlated with the overall proportion of spermatozoa with noncondensed chromatin, an individual spermatozoon's morphology appeared to be closely related to its chromatin condensation status. Morphologically normal spermatozoa with noncondensed chromatin were seen in all patients; the proportion averaged 23.3% [min 10.9%-max 44.4%]. Morphologically abnormal spermatozoa were more likely to have noncondensed chromatin than morphologically normal ones (P < 0.0001). Small-, large- or multiple-headed spermatozoa presented the highest degree of noncondensation (>80% for each type), and more than half the vacuolated spermatozoa also presented noncondensed chromatin. However, a morphologically normal spermatozoon may also have a noncondensed chromatin.

Syncytin-1 and its receptor is present in human gametes.

MAIN PURPOSE AND RESEARCH QUESTION: To determine whether the true fusogen Syncytin-1 and its receptor (ASCT-2) is present in human gametes using qRT-PCR, immunoblotting and immunofluorescence. METHODS: Donated oocytes and spermatozoa, originating from a fertility center in tertiary referral university hospital, underwent qRT-PCR, immunoblotting and immunofluorescence analyzes. RESULTS: Quantitative RT-PCR of sperm samples from sperm donors showed that syncytin-1 is present in all samples, however, protein levels varied between donors. Syncytin-1 immunoreactivity predominates in the sperm head and around the equatorial segment. The receptor ASCT-2 is expressed in the acrosomal region and in the sperm tail. Moreover, ASCT-2, but not syncytin-1, is expressed in oocytes and the mRNA level increases with increasing maturity of the oocytes. CONCLUSIONS: Syncytin and its receptor are present in human gametes and localization and temporal appearance is consistent with a possible role in fusion between oocyte and sperm.

Paternal exposure to testis cancer chemotherapeutics alters sperm fertilizing capacity and affects gene expression in the eight-cell stage rat embryo.

Treatment of testicular cancer includes the coadministration of bleomycin, etoposide and cis-platinum (BEP); however, along with its therapeutic benefit, BEP exposure results in extensive reproductive chemotoxic effects, including alterations to sperm chromatin integrity. As an intact paternal genome is essential for successful fertilization and embryogenesis, we assessed the effect of paternal exposure to BEP on sperm fertilization capacity and the resulting consequences on early embryonic gene expression. Adult male Brown Norway rats received a 9-week treatment with BEP or saline and then were sacrificed immediately or subject to a 9-week recovery period. HSP90AA1, HSP90B1 and PDIA3, involved in spermatozoa-egg interactions, were overexpressed in BEP-exposed spermatozoa after the 9-week treatment period; overexpression was also observed in spermatozoa from BEP-treated rats after 9 weeks of recovery. These proteins were localized to the plasma membrane of the sperm head; this localization may facilitate their role in spermatozoa-egg interactions as the highest staining intensities were observed in capacitated spermatozoa. The fertilization potential of spermatozoa was determined by in vitro fertilization with oocytes from unexposed naturally cycling female rats. Interestingly, the fertilization potential of spermatozoa following a 9-week recovery period from BEP treatment was significantly enhanced compared with controls. Moreover, stem cell transcription factors, involved in the regulation of a plethora of early embryonic events, were upregulated by more than twofold in eight-cell stage embryos sired by BEP recovery males compared with controls; this suggests that there are potential deleterious effects on embryo development well after termination of BEP exposure.

Expression and functional activity of PACAP and its receptors on cumulus cells: effects on oocyte maturation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1-R (PACAP type 1 receptor) are transiently expressed in granulosa cells (GCs) of mouse preovulatory follicles and affect several parameters associated with the ovulatory process. We investigated the expression of PACAP and its receptors in cumulus cells (CCs) after the LH surge and their role on cumulus expansion/apoptosis and oocyte maturation. PACAP and PAC1-R expression increased in CCs isolated at different times after treatment with human chorionic gonadotropin (hCG). Moreover, PACAP was able to reverse the inhibition of oocyte meiotic maturation caused by hypoxantine in cumulus cell-oocyte complexes (COCs) and efficiently promoted male pronuclear formation after fertilisation. PACAP was also able to induce cumulus expansion and prevent CC apoptosis. Our results demonstrated the induction of PACAP and its receptors in CCs by LH and EGF, suggesting that PACAP may play a significant role in the complex interactions of gonadotropin and growth factors during ovulation and fertilisation.

Frozen-thawed rhesus sperm retain normal morphology and highly progressive motility but exhibit sharply reduced efficiency in penetrating cervical mucus and hyaluronic acid gel.

The preservation of the genetic diversity of captive populations of rhesus monkeys is critical to the future of biomedical research. Cryopreservation of rhesus macaque sperm is relatively simple to perform, yields high post-thaw motility, and theoretically, provides via artificial insemination (AI) a way to easily transfer genetics among colonies of animals. In the interest of optimizing semen cryopreservation methods for use with vaginal AI, we evaluated the ability of frozen-thawed rhesus sperm to penetrate periovulatory cervical mucus (CM). Motile sperm concentration of pre-freeze ("fresh") and post-thawed ("thawed") samples from five different males were normalized for both computer assisted sperm motion analysis and CM penetration experiments. Sperm samples were deposited into slide chambers containing CM or gel composed of hyaluronic acid (HA) as a surrogate for CM and numbers of sperm were recorded as they entered a video field a preset distance from the sperm suspension-CM (or HA) interface. Fresh and thawed sperm were dried on glass slides, "Pap"-stained, and assessed for changes in head dimensions and head and flagellar shape. While retaining better than 80% of fresh sperm progressive motility, thawed sperm from the same ejaculate retained on average only 18.6% of the CM penetration ability. Experiments using HA gel yielded similar results only with reduced experimental error and thus improved detection of treatment differences. Neither the percentage of abnormal forms nor head dimensions differed between fresh and thawed sperm. While findings suggests that sperm-CM interaction is a prominent factor in previous failures of vaginal AI with cryopreserved macaque sperm, neither sperm motility nor morphology appears to account for changes in the ability of cryopreserved sperm to penetrate CM. Our data points to a previously unidentified manifestation of cryodamage which may have implications for assessment of sperm function beyond the cervix and across mammalian species.

Can intracytoplasmic morphologically selected sperm injection be used to select normal-sized sperm heads in infertile patients with macrocephalic sperm head syndrome?

OBJECTIVE: To study the chromosomal content of spermatozoa selected by intracytoplasmic morphologically selected sperm injection (IMSI) in cases of macrocephalic sperm head syndrome. DESIGN: Case report. SETTING: Obstetrics, gynecology, urology, and reproductive biology departments. PATIENT(S): Two infertile patients with large-headed spermatozoa. INTERVENTION(S): Fluorescence in situ hybridization on selected spermatozoa with normal-sized heads after IMSI selection. MAIN OUTCOME MEASURE(S): Percentages of polyploid, diploid, haploid aneuploid, and normal spermatozoa. RESULT(S): Of the six spermatozoa that could be selected, all were haploid but aneuploid. CONCLUSION(S): Absence of normal haploid spermatozoa among high magnification-selected spermatozoa contraindicated IMSI for these two patients.

Rnf19a, a ubiquitin protein ligase, and Psmc3, a component of the 26S proteasome, tether to the acrosome membranes and the head-tail coupling apparatus during rat spermatid development.

We report the cDNA cloning of rat testis Rnf19a, a ubiquitin protein ligase, and show 98% and 93% protein sequence identity of testicular mouse and human Rnf19a, respectively. Rnf19a interacts with Psmc3, a protein component of the 19S regulatory cap of the 26S proteasome. During spermatid development, Rnf19a and Psmc3 are initially found in Golgi-derived proacrosomal vesicles. Later on, Rnf19a, Psmc3, and ubiquitin are seen along the cytosolic side of the acrosomal membranes and the acroplaxome, a cytoskeletal plate linking the acrosome to the spermatid nuclear envelope. Rnf19a and Psmc3 accumulate at the acroplaxome marginal ring-manchette perinuclear ring region during spermatid head shaping and in the developing sperm head-tail coupling apparatus and tail. Rnf19a and Psmc3 may interact directly or indirectly with each other, presumably pointing to the participation of the ubiquitin-proteasome system in acrosome biogenesis, spermatid head shaping, and development of the head-tail coupling apparatus and tail.

Removal of acrosomal membrane from sperm head improves development of rat zygotes derived from intracytoplasmic sperm injection.

When intracytoplasmic sperm injection (ICSI) is applied in the rat, sperm chromatin is introduced into the oocyte together with the acrosome, which does not enter the cytoplasm of the oocyte during normal fertilization, resulting in the rat giving birth to pups. Since successful ICSI was reported in rats, but with low efficiency, it has been observed that the acrosome of the sperm head seems to have detrimental effects on the embryonic development of ICSI oocytes. To improve ICSI in rats, the effects of removal of the acrosomal membrane from rat sperm on the development of ICSI oocytes were examined. While most control (non-treated) sperm had an intact acrosomal membrane, the Triton X-100 (TX)- and lysolecithin (LL)-treated groups showed high percentages of sperm with a removed acrosomal membrane. The timing of pronuclear formation in ICSI-oocytes using TX- or LL-treated sperm was significantly accelerated compared with that of the control sperm (P<0.05). However, neither TX nor LL treatment affected amounts of PLCzeta in rat sperm. The rates of offspring derived from TX- (20.3 +/- 4.4%) and LL-treated sperm (19.0 +/- 2.8%) were also significantly higher than that of the control group (7.6 +/- 2.3%; P<0.05). Our data clearly indicate that removal of acrosomal membranes from sperm by reagents is effective for generation of offspring via ICSI in rats.

Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.

Heat-shock factor 1 (HSF1) protects cells and organisms against various types of stress, either by triggering a complex response that promotes cell survival or by triggering cell death when stress-induced alterations cannot be rescued. Although this dual role of HSF1 was observed in spermatogenesis exposed to heat shock or proteotoxic stress, HSF1 was also reported to contribute to cell resistance against genotoxic stress, such as that caused by doxorubicin, an anticancer drug in common clinical use. To better understand the stress/cell-dependent functions of HSF1, we used wild-type and Hsf1(tm1Ijb)/Hsf1(tm1Ijb) males to determine the role of HSF1 in the genotoxic stress response elicited in spermatogenic cells. Within 2 days after a single intraperitoneal injection of doxorubicin (DOXO; 5 mg/kg), proliferation of Hsf1+/+ but not Hsf1-/- spermatogenic cells was significantly reduced, whereas cell death was increased in mitotic germ cells and metaphase I spermatocytes. By 21 days, meiotic cells were depleted in all treated Hsf1+/+ testes but not in Hsf1-/- ones. Nevertheless, after 3 mo, spermatogenesis showed better signs of recovery in Hsf1+/+ than in Hsf1-/- males. Taken together, these data indicate that acute response to genotoxic stress in the testis involves HSF1-dependent mechanisms that induce apoptotic cell death in a TRP53-independent manner, but also intervene on a longer term to restore seminiferous tubules.

Factors affecting chromatin stability of bovine spermatozoa.

The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertilization process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on incidence of sperm nuclear chromatin instability (NCI), and (2) effects of the interaction between variation of NCI within a frozen ejaculate and variation of oocytes quality due to maturation time and/or season on the efficiency of in vitro embryo production (IVEP). Semen samples were collected once a week from six bulls using an AV and only ejaculates (n=220) of >0.30x10(9) sperm/ml and >or=60% motility were used. NCI was measured by: (1) detection of lysine-rich histones in sperm chromatin using aniline blue staining, (2) sperm susceptibility to acid-induced nuclear DNA denaturation in situ using acridine orange test, and (3) sperm susceptibility to nuclear chromatin decondensation (NCD). Bovine oocytes (n=695) were matured in vitro for 18 or 24 h, fertilized after sperm selection through a swim-up procedure and cultured for 72 h. The results showed that the 2nd ejaculates were superior to the 1st ones with respect to chromatin stability. Dilution of semen to 49.67+/-8.56x10(6) sperm/ml (1:19) decreased resistance of sperm to NCD. Cooling of semen had no significant effect on chromatin stability. Cryopreservation of semen augmented sperm vulnerability to DNA denaturation. Improvement of SQC (semen volume, sperm motility, velocity, viability and morphological normalcy) was generally concomitant with increase of sperm resistance to NCI. While Blonde d'Aquitaine bulls had a resistance to NCD higher than Limousine bulls in fresh semen, the former showed a greater susceptibility to DNA denaturation than the latter in cooled semen. Individuality significantly influenced NCI. The variability of NCI within a frozen ejaculate affected efficiency of IVEP. Significant negative correlations were observed between incidence of NCI and both fertilization rate and developmental capacity of embryos after maturation of oocytes for 18 h. The significant variation in IVEP traits due to season was independent of the effect of sperm chromatin instability.

Impaired hyperactivation of human sperm in patients with infertility.

Hyperactivation and acrosome reaction are prerequisite steps for sperm to be able to fertilize an oocyte. In mammals, hyperactivation is defined as a movement pattern seen in spermatozoa at the site and time of fertilization. The objectives of the present experiments were to analyze the process of hyperactivation and to investigate its relationship with progesterone evoked intracellular calcium concentration ([Ca2+]i) increase and their implications with infertility. After capacitation the sperm from patients, when compared with donor's sperm, showed decreased percentage of hyperactivated sperm, molitily, progressive motility, and curvilinear velocity (VCL). On the other hand, the linearity (LIN) was increased. Amplitude of lateral head displacement (ALH) and [Ca2+]i increase (peak and plateau from baseline) showed good correlation in patients with infertility. These data suggest that impaired hyperactivation might be involved in the pathophysiology of infertility.

The partial head decondensation test is a new, quick method to assess acrosome status in human spermatozoa.

OBJECTIVE: To develop a fast method for assessing acrosome status in human spermatozoa. DESIGN: Development of a new in vitro test to assess acrosome reaction in human spermatozoa. SETTING: Academic medical institution. PATIENT(S): Normozoospermic subjects. INTERVENTION(S): Spermatozoa were isolated from fresh semen samples, capacitated, and stimulated or not with P or ionomycin. Acrosome reactions were evaluated by phase-contrast microscopy after a brief sperm incubation in a decondensing solution. The results were compared with those obtained by scanning electron microscopy and fluoresceinated lectin staining. MAIN OUTCOME MEASURE(S): Percentage of intact acrosomes. RESULT(S): The new procedure allowed intact acrosomes to be easily identified and quantified by phase-contrast microscopy. In unstimulated and ionomycin-treated spermatozoa, a very good agreement was found among the new test, scanning electron microscopy, and fluoresceinated lectin staining. In P-treated spermatozoa, the proposed method allowed a significantly higher percentage of reacted acrosomes to be resolved, likely due to its ability to detect the very initial stages of the acrosome reaction. CONCLUSION(S): The new test allows acrosome-intact and acrosome-reacted spermatozoa to be unambiguously singled out and quantified. The method is rapid, reliable, sensitive, and easy to perform, which makes it of profitable use in both basic research and diagnostic practice.

Effects on in vitro embryo development and intracellular glutathione content of the presence of thiol compounds during maturation of prepubertal goat oocytes.

The low number of embryos produced from in vitro matured, fertilized, and cultured (IVM-IVF-IVC) oocytes of prepubertal goat is mainly due to a low incidence of sperm head decondensation at fertilization (Martino et al., 1995: Theriogenology 43:473-485; Mogas et al., 1997: Theriogenology 48:815-829). Thiol compounds stimulate glutathione (GSH) synthesis and improve the rates of male pronucleus (MPN) formation and embryo development. The present study was carried out to determine whether supplementation of the IVM medium with 100 microM of cysteamine, 100 microM of beta-mercaptoethanol, 0.57 mM of cysteine, and 0.57 mM cystine might improve the embryo development and intracellular GSH level of prepubertal goat oocytes. After 27 hr post IVM, a sample of oocytes was frozen and the intracytoplasmic GSH content was evaluated by spectrophotometry. IVM-oocytes were inseminated with fresh semen and cultured in SOF medium. Only the addition of cysteamine to IVM media significantly improved the percentage of the morula plus blastocyst yield compared to the control group (oocytes matured in absence of thiol compounds) (22.2 vs. 6.4%, respectively; P < 0.05). The percentage of expanded blastocysts in cysteamine and control groups was 13.0 and 2.6%, respectively, and the mean cell number per blastocyst was 86.8 and 60.5, respectively. None of the other thiol compounds studied significantly improved the percentage of embryos obtained. It has been demonstrated that prepubertal goat oocytes synthesize GSH during IVM and that thiol compounds increase this GSH synthesis. In conclusion, only the addition of 100 microM of cysteamine to the maturation medium improves embryo development from prepubertal goat oocytes although all the thiol compounds used in this study increased intracellular GSH content.

Involvement of cytoplasmic free calcium in boar sperm: head-to-head agglutination induced by a cell-permeable cyclic adenosine monophosphate analog.

When boar spermatozoa are incubated in a medium designed for in vitro fertilization, many of them become agglutinated at the acrosomes. We previously reported that bicarbonate and cyclic adenosine 3',5'-monophosphate (cAMP) promote agglutination. The aim of the present study is to examine the role of cytoplasmic free Ca(2+) in boar sperm agglutination induced by a cell-permeable cAMP analogue. Spermatozoa were collected from five mature boars, washed, and resuspended in a modified Krebs-Ringer-Hepes solution lacking calcium chloride. The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 minutes and were then used to determine the percentages of head-to-head agglutinated spermatozoa. Percentages of head-to-head agglutinated spermatozoa in the samples rose significantly after incubation, from 28% to 61%-62%, after adding to the medium a cell-permeable, phosphodiesterase-resistant cAMP analogue (cBiMPS, 10 microM) or an adenylyl cyclase stimulator (sodium bicarbonate, 5 mM) plus a cell-permeable phosphodiesterase inhibitor (IBMX, 25 microM). However, the promoting effects of these reagents were blocked when spermatozoa were pretreated with a cell-permeable Ca(2+) chelator (BAPTA-AM, 25 microM), whereas the same pretreatment with a cell-impermeable Ca(2+) chelator (BAPTA, 25 microM) had almost no influence on sperm agglutination. Adding thapsigargin, a potential Ca(2+)-ATPase inhibitor, to the medium raised the percentages of agglutinated spermatozoa in a concentration-dependent manner for concentrations up to 4 microM. When 4 microM thapsigargin and 10 microM cBiMPS were examined for their effects on free Ca(2+) levels in sperm heads by using a cell-permeable Ca(2+) indicator (fluo-3/AM), the samples incubated with both or either of these reagents contained many head-to-head agglutinated cells that exhibited intense fluorescence in the heads. In control samples incubated without these reagents by contrast, most spermatozoa were free (unagglutinated) cells and characterized by almost no or only slight fluorescence in the heads. Moreover, morphological observation of Giemsa-stained preparations revealed that most agglutinated spermatozoa possessed darkly stained acrosomes, which distinguished them from acrosomereacted spermatozoa. This indicated that the sperm agglutination was not a result of the acrosome reaction. Furthermore, with indirect immunofluorescence of Ca(2+)-ATPases, the mouse monoclonal antibody to this enzyme demonstrated high affinity to the acrosomes of permeabilized spermatozoa. Based on these results, we conclude that cytoplasmic free Ca(2+) is involved in sperm head-to-head agglutination induced by a cAMP analogue.

Fertilization and embryo development in a mouse ICSI model using human and mouse sperm after immobilization in polyvinylpyrrolidone.

BACKGROUND: For human ICSI, sperm are normally immobilized immediately prior to injection. However, there are some situations when only sperm of questionable viability are available. There are few evaluations of fertilization or developmental problems in human or animal models using sperm having known intervals between immobilization and injection. METHODS: Immobilized human sperm were maintained for 1-24 h in 10% polyvinylpyrrolidone (PVP) before injection into mouse oocytes. Mouse sperm heads were similarly maintained in either PVP or a high potassium-containing 'nucleus isolation medium' (NIM) before ICSI and embryo development to the blastocyst stage. RESULTS: Immobilized human sperm activated mouse oocytes comparably to controls even 24 h after immobilization. However, mouse sperm heads showed a decrease in activating ability 6 h after isolation, either in PVP or NIM. A significant reduction in blastocyst development occurred if mouse sperm heads were maintained for even 1 h in PVP. After 6 h, no blastocysts formed, with arrest occurring at the morula stage. NIM provided partial protection for up to 3 h. CONCLUSIONS: Immobilized human sperm maintained oocyte activating activity for 24 h. However, mouse sperm are susceptible to alterations that affect both fertilization and development.