Rat Sperm Heads Research Articles

Membrane Restructuring Events during the Enzymatic Generation of Ceramides with Very Long-Chain Polyunsaturated Fatty Acids.

In rat sperm heads, sphingomyelin (SM) species that contain very long-chain polyunsaturated fatty acid (V-SM) become ceramides (V-Cer) after inducing in vitro the acrosomal reaction. The reason for such a specific location of this conversion, catalyzed by a sphingomyelinase (SMase), has received little investigation so far. Here, the effects of SMase were compared in unilamellar vesicles (large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs)) containing phosphatidylcholine, and either V-SM or a palmitate-rich SM (P-SM). In uniformly sized LUVs at 37 °C, more V-Cer was generated and more rapidly than P-Cer. Nephelometry and dynamic light scattering showed that LUVs tended to form large lipid particles more intensely, and Förster resonance energy transfer (FRET) increases suggested that lateral lipid mixing was more marked when V-Cer rather than P-Cer was produced. As reported by 6-dodecanoyl-2-dimethyl-aminopnaphthalene (Laurdan) and 1,6-diphenyl-1,3,5,-hexatriene (DPH), the production of V-Cer resulted in higher and faster restriction in lipid mobility than that of P-Cer, implying a stronger increase in membrane dehydration and microviscosity. Moreover, DPH anisotropy suggested a higher solubility of V-Cer than that of P-Cer in the liquid-disordered phase. At room temperature, liquid-condensed lateral domains appeared in P-SM- but not in V-SM-containing GUVs. The former maintained their size while losing their contents gradually during SMase action, whereas the latter became permeable earlier and reduced their size in few minutes until suddenly collapsing. The fast and potent generation of V-Cer may contribute to the membrane restructuring events that occur on the acrosome-reacted sperm head.

High-quality human and rat spermatozoal RNA isolation for functional genomic studies.

Sperm RNA is a sensitive monitoring endpoint for male reproductive toxicants, and a potential biomarker to assess male infertility and sperm quality. However, isolation of sperm RNA is a challenging procedure due to the heterogeneous population of cells present in the ejaculate, the low yield of RNA per spermatozoon, and the absence of 18S and 28S ribosomal RNA subunits. The unique biology of spermatozoa has created some uncertainty in the field about RNA isolation methods, indicating the need for rigorous quality control checks to ensure reproducibility of data generated from sperm RNA. Therefore, we developed a reliable and effective protocol for RNA isolation from rat and human spermatozoa that delivers highly purified and intact RNA, verified using RNA-specific electrophoretic chips and molecular biology approaches such as RT-PCR and Western blot analysis. The sperm RNA isolation technique was optimized using rat spermatozoa and then adapted to human spermatozoa. Three steps in the sperm isolation procedure, epididymal fluid collection, sperm purification, and spermatozoon RNA extraction, were evaluated and assessed. The sperm RNA extraction methodology consists of collection of rat epididymal fluid with repeated needle punctures of the epididymis, somatic cell elimination using detergent-based somatic cell lysis buffer (SCLB) and the use of RNA isolation Kit. Rat sperm heads are more resistant to disruption than human spermatozoa, necessitating the addition of mechanical lysis with microbeads and heat in the rat protocol, whereas the human sperm protocol only required lysis buffer. In conclusion, this methodology results in reliable and consistent isolation of high-quality sperm RNA. Using this technique will aid in translation of data collected from animal models, and reproducibility of clinical assessment of male factor fertility using RNA molecular biomarkers.

SpermBase: A Database for Sperm-Borne RNA Contents.

Since their discovery approximately three decades ago, sperm-borne RNAs, both large/small and coding/noncoding, have been reported in multiple organisms, and some have been implicated in spermatogenesis, early development, and epigenetic inheritance. Despite these advances, isolation, quantification, and annotation of sperm-borne RNAs remain nontrivial. The yields and subspecies of sperm-borne RNAs isolated from sperm can vary drastically depending on the methods used, and no cross-species analyses of sperm RNA contents have ever been conducted using a standardized sperm RNA isolation protocol. To address these issues, we developed a simple RNA isolation method that is applicable to sperm of various species, thus allowing for reliable interspecies comparisons. Based on RNA-Seq analyses, we established SpermBase ( www.spermbase.org), a database dedicated to sperm-borne RNA profiling of multiple species. Currently, SpermBase contains large and small RNA expression data for mouse, rat, rabbit, and human total sperm and sperm heads. By analyzing large and small RNAs for conserved features, we found that many sperm-borne RNA species were conserved across all four species analyzed, and among the conserved small RNAs, sperm-borne tRNA-derived small noncoding RNAs and miRNAs can target a large number of genes known to be critical for early development.

Efectos toxicológicos del uso prolongado e intenso de emisiones de espirales contra mosquitos en ratas y sus implicaciones sobre el control de la malaria

Mosquito coil is a vector control option used to prevent malaria in low income counties, while some studies have addressed this issue, additional reseach is required to increase knowledge on the adverse health effects caused by the prolonged use of coils. In this study we investigated the toxicological effects of fumes from two locally manufactured mosquito coil insecticides (with pyrethroids: transfluthrin and d-allethrin as active ingredients) on male albino rats. For this, we recorded the haematological and biochemical indices, and made histopathology and mutagenicity evaluations in rats exposed to mosquito fumes during 2, 4, 8, 12 and 16 week periods. Haematological determination was performed using automated hematology analyzer to determine White Blood Cell (WBC), Packed Cell Volume (PCV), Red Blood Cell (RBC) and Platelet (PLT) counts, while biochemical evaluations were determined using available commercial kits. Gross histopathological changes were studied for the kidney, liver and lungs in sacrificed rats. The rat sperm head abnormalities assessment was used to evaluate mutagenicity. Mosquito coil fumes produced significant increase (P < 0.05) in the levels of total protein, total albumin and bilirubin, when animals were exposed from two weeks to 16 weeks with transfluthrin. Similarly, elevation in the activities of aspartate amino transferase, alanine amino transferase and alanine phosphatase, increased significantly in both insecticides. Increase in WBC, RBC and PCV were recorded for all the exposure periods, however PLT count showed no significant increase (P > 0.05). Mutagenicity assessment revealed sperm abnormality was statistically significant (P < 0.05) compared with the control at 8, 12 and 16 weeks post exposure to transfluthrin. Histological studies revealed severe lung damage evidenced by interstitial accumulations, pulmonary oedema and emphysema in exposed rats. Intracellular accumulations and severe sinusoidal congestion of liver cells were observed from 12 weeks exposure, indicating liver damage. Our studies indicate that mosquito coil fumes do initiate gradual damage to the host. These pathological effects must be taken into consideration by the malaria control program, particularly when regulating their long term and indoor usage.

Exposure to Bleomycin, Etoposide, and Cis-Platinum Alters Rat Sperm Chromatin Integrity and Sperm Head Protein Profile1

Testicular cancer, currently the most common cancer affecting men of reproductive age, is one of the most curable malignancies due to the progress made in the early diagnosis and effective treatment of this disease. The coadministration of bleomycin, etoposide, and cis-platinum (BEP) has brought the 5-yr survival rate of testis cancer patients to over 90%. However, this treatment results in reproductive chemotoxic effects. We assessed the effect of BEP treatment on sperm chromatin integrity and sperm head protein profiles of adult male Brown Norway rats following 9 wk of treatment with BEP and in animals treated for 9 wk and then subjected to a 9-wk recovery period. Both the susceptibility of DNA to denaturation and the number of strand breaks were significantly increased in mature sperm following 9 wk of treatment with BEP; proteomic analysis revealed that the expression of several proteins, including HSP90AA1 and HSP90B1, was markedly affected. Following a 9-wk recovery period, mature sperm did not show significant DNA damage, indicating that repair had potentially occurred. Interestingly, the protamination level of the sperm of these animals was significantly decreased, while histones HIST1H1D (H1.2), HIST1H4B (H4), HIST2H2AA3 (H2A1), and HIST1H2BA (H2B1A) were concomitantly up-regulated; this was not observed in the sperm immediately following 9 wk of treatment. Thus, there are persistent effects on proteins in sperm heads from the cauda epididymidis 9 wk posttreatment, in the absence of DNA strand breaks. We suggest that these effects on the sperm head proteome may contribute to long-lasting adverse effects in the progeny of BEP-exposed males.

Sprague Dawley rat sperm classification using hybrid multilayered Perceptron network

As of now, the analysis of sperm such as counting and detection processes are still operated manually which tends to produce errors. False detection in sperm analysis must be minimized as possible. Therefore, the current study focuses on developing a Sprague Dawley rat sperm classification system to assist the detection process by pathologist. The system has the ability to classify the Sprague Dawley rat sperm into two classes namely normal and abnormal based on the morphological characteristic of sperm's head. The proposed system employs the Hybrid Multilayered Perceptron (HMLP) neural network trained with the Modified Recursive Prediction Error (MRPE) algorithm as the intelligent classifier tool. This study will also investigate three significant morphological characteristics of rat sperm's head namely opened degree, width of the curve and template matching percentage as the input data for the HMLP network. Furthermore, this study further classifies the abnormal sperm into hookless and banana shape. Promising result with 100% and 94.62% of accuracy has been achieved for two classes and three classes classification of rat sperm respectively.

273 REMOVAL OF ACROSOME FROM SPERM HEADS IMPROVES DEVELOPMENT OF RAT ZYGOTES THROUGH INTRACYTOPLASMIC SPERM INJECTION

In intracytoplasmic sperm injection (ICSI), sperm chromatin is introduced into the oocyte together with acrosome, which does not enter cytoplasm of oocytes during normal fertilization. In mice, acrosome of sperm head has a detrimental effect on embryonic development of the ICSI oocytes (Morozumi et al. 2006 Proc. Natl. Acad. Sci. USA 103, 17 661–17 666). We examined the effect of acrosome removal of frozen/thawed (F/T) rat sperm on development to term of the ICSI oocytes in order to improve production efficiency of live offspring from F/T rat spermatozoa through ICSI. In experiment 1, epididymal spermatozoa of the Wistar rats were frozen as described previously (Seita et al. 2005 Reprod. Fertil. Dev. 18, 256 abst). The F/T spermatozoa were sonicated to separate sperm heads. The sperm heads were divided into 3 treatment groups; nontreated sperm head (control), sperm heads exposed in 0.02% of Triton X-100 solution for 1 min (TX), sperm heads exposed in 0.02% lysolectin solution for 1 min (LL). Acrosomal status of the sperm heads was then examined by FITC-peanut agglutinin stain. In experiment 2, sperm heads of the control, TX, and LL treatments were microinjected into denuded oocytes obtained from superovulated females (Hirabayashi et al. 2002 Transgenic Res. 11, 221–228). The ICSI oocytes were cultured and observed for the formation of pronuclei (2PN) for 6 h and blastocyst formation at 120 h of culture. In experiment 3, the ICSI oocytes cultured for 6 h were transferred to recipient females to examine development to term. Statistical analyses of the results were carried out by 1-way ANOVA. In experiment 1, the TX (76%) and LL (89%) treatments showed higher rates of acrosome removal than that of the control (24%) group (P &lt; 0.05). In experiment 2, the percentage of 2PN formation at 6 h after ICSI was not significantly different among sperm-treated groups, although the TX (76%) and LL (70%) groups were higher than the control (24%) group at 4 h (P &lt; 0.05). The percentages of blastocyst formation were not significantly different among sperm-treated groups (control: 10%; TX: 25%; and LL: 25%). In experiment 3, the efficiency of development to term of the TX treatment group (21%: 12/57) was significantly higher than the control (5%: 3/55; P &lt; 0.05), although the LL treatment group (16%: 12/75) was not significantly different from the control and TX treatment groups. These results indicate that acrosome removal using TX of F/T rat sperm heads before ICSI improves development to term of the ICSI oocytes. This benefical effect of acrosome removal may be due to the quick release of sperm-activating factors from sperm heads after ICSI in the rat.

Ultrastructural observations on the Sperm of two Apodemus species,Apodemus agrarius coreae and Apodemus speciosus peninsulae,in Korea

Sperm morphology of Apodemus agrarius coreae, Apodemus speciosus peninsulae, Iaboratory mouse and rat were studied. The total length of the sperm ofA. a. coreae, A. s. peninsulae, the mouse and rat was about 133lhm, 124lhm, 13l~em and 192;hm, respectively. Two Apodemus spenu heads had a fish hook shape, while the mouse sperm head had a hook shape; thus, the sperm heads of genus Apodemus were somewhat like those of the mouse. The rat sperm head was unlike the other species in that it had a falciform shape. In addition, two Apodemus spermatozoa had two well-developed ventral spurs (Vs), and the mouse sperm head also had two sirnilar small Vs, but rat sperm head had low ridge line-like Vs. All species had a separated head-cap segment in the ventral side of the anterior acrosomal portion of sperm. In the neck region, the vault formerly occupied by the proxirnal centriole was retained in a niche in the dense substance of the connecting piece in the spermatozoa of two Apodemus species, the mouse and the rat. The neck region of two Apodemus spermatozoa was surrounded by the scroll of a redundant nuclear envelope and a few large mitochondoria. In contrast, in the mouse and rat spermatozoa, the neck region was devoid of mitochondoria, except a fold of the redundant nuclear envelope. Since a very similar sperm head morphology occurs in the two Apodemus species and the mouse, it suggests that these genera are sister groups, to the exclusion of the rat.

Offspring Derived from Intracytoplasmic Injection of Sonicated Rat Sperm Heads

ABSTRACT The present study investigated the effect of separation of spermatozoa by sonication or Piezo-pulse on in vitro development of oocytes injected with sperm heads in the rat. We also examined development to term of rat oocytes injected with sperm heads. Rat frozen-thawed spermatozoa were separated into heads and tails by sonication for 10 sec or Piezo-pulse in KRB medium, and each treated sperm head was injected into an ooplasm. The oocytes were observed for formation of two pronuclei and development to 2-cell embryos. The percentages of formation of two pronuclei and development to the 2-cell stage did not significantly (P>0.05) differ between the two groups. Oocytes injected with sonicated sperm heads that reached the pronuclear stage at 10 h after injection of sperm heads were transferred into 7 recipients. Five recipients became pregnant, and 8 living pups were obtained. The results indicate that rat oocytes injected with sonicated sperm heads can develop to term in vivo. Furthermore, no differ...

Cloning of rat sp56, the homologue of mouse sperm ZP3 receptor-sp56.

Mouse sp56 is considered as one of the candidates for mouse zona pellucida 3 (mZP3) receptor. Up to date, its homologue has only been cloned from guinea pig, namely AM67. Based on the cDNA sequence of mouse sp56, we designed a pair of primer to amplify its homologue from rat testis cDNA. Using RT-PCR, two fragments of 743 bp and 938 bp were amplified. The PCR products show very high homology to mouse sp56. However, the 743 bp product completely lacks one of the seven Sushi domains of mouse sp56. Using the 743 bp product as the probe to detect the expression profile of sp56 in rat tissues, Northern blot shows that a approximately 2.0 kb mRNA expresses specifically in testis. Employed the RACE method, two full cDNA sequences of rat sp56 were obtained. A Mr approximately 42 KD band was detected in denatured and non-reducing protein sample of rat testis and sperm with anti-mouse sp56 monoclonal antibody by Western blot method. Rat sp56 was localized on rat sperm head by the indirect immunofluorescence method. Rat sp56 immunoreactivity was detected from the early pachytene spermatocytes and throughout the spermatogenesis. Its cloning will further our understanding of the mechanism of the sperm-egg recognition and binding.

Development of rat oocytes following intracytoplasmic injection of sperm heads isolated from testicular and epididymal spermatozoa

The possibility of obtaining normal development of rat oocytes following intracytoplasmic injection of rat sperm heads, obtained by sonicating spermatozoa from testes and epididymides, was evaluated. Irrespective of the source of spermatozoa, sperm heads were successfully injected into ∼45% of oocytes used; after 9–12 h of culture, ∼55% of injected oocytes still had normal morphology. Of the oocytes injected with testicular sperm heads 45% were activated, with a female pronucleus and a second polar body, but significantly more oocytes (∼68%) injected with caput and cauda epididymal sperm heads were activated. Male pronuclear formation was observed in 67–84% of the activated oocytes, with no difference in the proportions among the different sources of sperm heads. When zygotes showing two pronuclei and a second polar body at 10 h after injection were cultured in conditions that support development of 1-cell embryos produced in vivo, no embryos derived from testicular sperm heads developed to blastocysts after 120 h of culture. Development of embryos derived from cauda sperm heads was significantly higher at all points of assessment, while embryos from caput sperm showed an intermediate degree of development, compared with embryos from testicular spermatozoa. However, similar proportions (2–4%) of 1-cell embryos derived from all three groups of sperm heads developed into normal offspring after transfer to foster mothers; of the limited number of offspring tested, all were fertile. These results demonstrate that sperm heads from all sources tested are similar in their ability to contribute to full development of normal, fertile offspring.

Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum alpha-L-fucosidase from rat testis and epididymal spermatozoa.

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum alpha-L-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for alpha-L-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the alpha-L-fucosidase activity was associated with the 48,000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of alpha-L-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH-activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm-egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm-egg interactions.

Monoclonal antibody against a 52 K sperm surface protein inhibits sperm-zona pellucida interactions in the rat.

A major regulatory site for species specificity of fertilization in mammals lies at the level of sperm binding to the zona pellucida. This implies a high degree of complementarity between gamete and receptor molecules. These molecules support species-specific interactions between sperm and oocyte that lead to gamete fusion (fertilization). We identified a rat sperm head surface antigen using an IgG1 monoclonal antibody (HD1) against rat sperm of epididymis cauda. By electron microscopy the antigen was shown to be present on the plasma membrane surface of the sperm dorsal head. One- and two-dimensional immunoblotting analysis of sperm proteins demonstrated that HD1 reacted only with a 52 K molecule with a pI ranging from 6.6 to 7.2. The 52 K protein was first detected in situ by indirect immunofluorescence and showed to be underlining few elongated spermatids in testis. However, when the sperm reached the epididymis caput, the antigen was seen to be expressed on the dorsal surface of spermatozoa head. A similar fluorescence reaction was detected on sperms in the epididymis corpus and cauda. The specific spermatozoa-zona pellucida interaction was inhibited in the presence of monoclonal antibody HD1 in a sperm binding assay of in vitro fertilization.

Immunocytochemical detection of actin and 53 kDa polypeptide in the epididymal spermatozoa of rat and mouse.

Presence of immunocytochemically detectable actin in the rat and mouse sperm head has been enigmatic for years. In this study, we demonstrate actin in the perinuclear theca and show that the detection of actin epitopes in the rat and mouse epididymal spermatozoa can effectively be enhanced by pre-extraction of sperm cells with SDS. The study with one monoclonal and one polyclonal anti-actin antibody was carried out at conventional and confocal fluorescence and electron microscope level, and by immunoblotting of proteins isolated from the head and tail fractions. In the head of the control methanol-acetone fixed rat spermatozoa, the polyclonal antibody gave a stronger immunostaining in the postacrosomal area and in the perforatorium than the monoclonal antibody. In the mouse sperm head, the monoclonal antibody labeled the ventral edge of the postacrosomal area and slightly the perforatorium, whereas the polyclonal antibody stained the entire perinuclear space. In the SDS-extracted spermatozoa, an intense postacrosomal and perforatorial labeling was obtained with both antibodies but, in particular in the rat spermatozoa, the middle lateral portion of the postacrosomal segment remained unlabeled. Sonication seemed to cause structural modifications which specifically impeded staining with the monoclonal antibody. Both antibodies detected actin in the basal plate and the monoclonal antibody in the neck. Amorphous matrix of the connecting piece showed immunogold labeling. In the tail, the monoclonal antibody recognized actin and a relatively basic 53 kDa polypeptide, whereas the polyclonal antibody reacted with several protein bands. SDS-soluble actin of the tail was addressed to the midpiece and the SDS-insoluble 53 kDa protein profoundly to the outer dense fibers of the principal piece. Intense labeling of actin in the SDS-extracted rat and mouse spermatozoa was presumably due to the generated demasking of actin epitopes embedded in the perinuclear cytoplasm. The results are important in confirming that actin in the rat and mouse sperm head is not lost during spermiogenesis but apparently contributes to the three-dimensional packing of the mature perinuclear cytoplasm. This study further demonstrates the importance of the methods used in sample preparation and advantages of confocal microscopy when attempting to detect cytoskeletal proteins which, as in spermatozoa, may occur in small quantities.

Automated analysis of toxicant-induced changes in rat sperm head morphometry

An automated sperm morphometry analysis (ASMA) instrument was developed to obtain measurements of toxicant-induced changes in rat sperm head morphometry. 1,3-dinitrobenzene (1,3-DNB), a testicular toxicant known to affect sperm parameters, was used. Twelve-week-old Sprague-Dawley rats were allocated to a control (C) and to two 1,3-DNB treatment groups (T1 = 15 mg/kg; T2 = 25 mg/kg). 1,3-DNB was administered as a single dose by gavage, and animals were sacrificed 22 days after exposure. Sperm were collected, and morphology smears were made by a standard method. One hundred sperm heads were digitized on each slide, and 8 metric measurements were automatically reported. All measurements tended to decrease in a dose-dependent manner with increasing doses of 1,3-DNB. All values for total width (W a) and interior width (W e) were significantly different from one another. Wa/L was significantly larger in the control than in T1 or T2, and symmetry (S = W b/W a) was significantly smaller in the control than in T1 or T2. Multivariate cluster analysis revealed three subpopulations that were also visually distinct. Subpopulation no. 1 was normal, based on published descriptions of normal rat sperm; subpopulation no. 2 was abnormal with a flattened curvature and a normal length; subpopulation no. 3 was abnormal with a foreshortened length and a flattened curvature. T1 and T2 contained significantly more sperm from subpopulation no. 2 and no. 3 than C(T1 = 22% and T2 = 34% vs. C = 8% by cluster analysis). C had 93% normal sperm, while the treatments had 78% and 66%, respectively. Based on these findings, we conclude that ASMA is an efficient and accurate method of sperm morphometry analysis that may improve our ability to detect sperm biomarkers of reproductive health.

Extractions reveal specific argentophilic proteins in rat and bull sperm heads

Silver-stainability (argentophilia) of cytoplasmic structures occurring in spermatids have been localized into the organizing perinuclear theca, but the biochemical nature and structural associations of these proteins with the cytoskeletal and membranous elements are unresolved and, therefore, were the aim of the present study. Light and electron microscopic analysis of the silver-stainability in the rat spermatids and spermatozoa was carried out in the intact testis tissue and epididymal spermatozoa and after their chemical and mechanical extraction. Correlation of argentophilia with specific proteins of rat and bovine spermatids and spermatozoa was investigated using a recently developed technique for silver nitrate staining of proteins on nitrocellulose. Sequential formation of the silver-stainable domains seemed to proceed from the argentophilic acrosomal ring. Various extractions indicated that argentophilia in the spermatids and spermatozoa was mainly associated with the perinuclear theca and to some extent to the plasma membrane. Hyamine-soluble extract from spermatozoa of rat and bull revealed only a single argentophilic protein of 130 kDa. Hyamine and SDS-soluble extracts of rat testis tissue contained an additional group of argentophilic polypeptides of lower molecular weight (115, 94, 36, 23, and 21 kDa). Reduction in the number of argentophilic proteins appears to be involved in a series of changes in the cyto-architecture of developing spermatids. Tentative cytoskeletal nature of argentophilic proteins remains to be identified. Nevertheless, they may have important physical relations with the higher-order organization of the sperm head cytoskeleton and overlying membranes.

Small nuclear ribonucleoprotein polypeptides in rat sperm

1. 1. Immunoblot analysis of rat sperm head proteins revealed the presence of polypeptides recognized by anti-Sm serum obtained from patients with systemic lupus erythematosus. 2. 2. Two of these polypeptides have molecular weights of 26,000 and 15,000 and they were identified as small nuclear ribonucleoprotein components present in other rat tissues. 3. 3. When the autoimmune serum was used in the immuno-gold procedure for electron microscopy, gold particles were found only on the sperm nucleus. 4. 4. The results indicate that some polypeptides of the small nuclear ribonucleoprotein complex are components of the rat sperm chromatin.

In vitro study on the increase of trypsin resistance in the rat testicular sperm head.

The alteration of trypsin resistance in rat sperm head was investigated after the sperm were incubated in the culture media (199-Earele, 1% BSA) containing the inhibitors of protein synthesis. The head of non-incubated testicular sperm was easily digested by trypsin (1 mg/ml) while the head of the incubated sperm showed resistance to trypsin digestion after 3 hr-incubation. Trypsin resistance in epididymal sperm head did not change after the sperm was incubated. Neither cycloheximide (100 micrograms/ml) nor chloramphenicol (100 micrograms/ml) affected the trypsin resistance in the testicular sperm head.

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: Relationship of the manchette and multiple band polypeptides

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.

Antigens on rat spermatozoa with a potential role in fertilization

Eight monoclonal antibodies (McAbs), directed against antigens on rat cauda epididymal spermatozoa, were tested for their capacity to interfere with fertilization in vitro as a means of identifying molecules with a potential role in sperm-egg recognition and fusion. Antigens recognized by the McAbs were visualized on live spermatozoa by indirect immunofluorescence (IIF) and characterized by immunoblotting. Five McAbs (designated 1B5, 2C4, 4B5, 5B1, and 8C4) recognized antigens specifically on the sperm acrosome and three (designated 2B1, 2D6, and 6B2) bound to the flagellum. Of the eight McAbs investigated, three (2B1, 2C4, and 6B2) were effective in blocking fertilization in vitro when added as culture supernatants to mixtures of sperm and eggs. McAb 6B2 was inhibitory due to its ability to agglutinate spermatozoa. McAbs 2B1 and 2C4 did not agglutinate capacitated spermatozoa, had no observable effect on motility, and yet blocked fertilization in a dose-dependent manner. McAb 2C4 did not give a reaction on immunoblots, but the 2B1 antigen was identified as an Mr 40 kD glycoprotein. McAb 2B1 appeared to block fertilization at the level of zona binding, whereas the effects of 2C4 were directed more against zona penetration and/or fusion with the vitellus. When sperm-egg complexes were stained with 2C4 or 2B1 McAbs and viewed by IIF, all spermatozoa that were attached to the zona showed fluorescence on the head. These results suggest that different antigens on the rat sperm head participate in different aspects of the fertilization process and that during capacitation there is either exposure of these antigens or else they migrate to their site of action from the flagellum.