Late Spermatids Research Articles

Molecular and cytogenetic evaluation for potential genotoxicity of hydrocortisone

ObjectiveTo assess the risk of hydrocortisone sodium succinate through different end points of genotoxicity. MethodsThe study examined the induction of chromosomal aberrations in bone marrow cells, morphological sperm abnormalities, the effect on dominant lethal gene and protein synthesis. Hydrocortisone was given intraperitoneally at three dose levels 26, 39 and 52 mg/kg body weight which was equivalent to the therapeutic doses in man. ResultsThe results showed that single dose treatment with different doses had no effect on chromosomal aberrations. The dose of 52 mg/kg body weight induced significant percentage of chromosomal aberrations in bone marrow cells after repeated treatment for 7 and 14 days. Significant effect of morphological sperm abnormalities was demonstrated only after treatment with the dose of 52 mg/kg body weight. For examining the dominant lethal mutation, male mice were injected with dose of 39 mg/kg body weight for 5 consecutive days. Mating between treated males and virgin untreated females were performed at different time intervals. The results showed that the percentage of fertile mating at 1–7 and 8–14 days reduced to 50% and 60% respectively compared with control group while no effect was recorded at 15–21 days. The percentage of dominant lethal mutation reached 0.32%, 4.4% and 0% in mating intervals respectively indicating pronounced effect of hydrocortisone at the interval 8–14 days which represented by the late spermatids. The results also showed that the repeated treatment with the dose of 52 mg/kg body weight inhibited protein synthesis which contributed to the cytotoxic effect of the drug. ConclusionsIt is concluded that long term treatment with large doses of hydrocortisone may have genotoxic effect.

The impact of chronic testicular inflammatory infiltration on spermatogenesis in azoospermic men, evidence-based pilot study

Abstract Introduction: The testes are immune privileged organs but they are subjected to acute and chronic inflammations that can impair spermatogenesis. Spermatogonial stem cells are less sensitive to injury, capable of self-renewal, and act as a reservoir that can restart impaired spermatogenesis. Objective: To study the association of chronic testicular inflammatory infiltration with an area of complete spermatogenesis in testicular biopsies of men with non-obstructive azoospermia (NOA). Design: Retrospective study. Patients and methods: Review of 109 archived Bouin fixed, paraffin embedded and hematoxylin and eosin stained open testicular biopsies of azoospermic men. Main outcome measure: The absence/presence (focal/widespread) of chronic interstitial inflammatory infiltration and absence/presence of late spermatids. Results: Obstructive picture was found in 25 (22.9%) whereas non-obstructive patterns were found in 84 (77.1%) of the reviewed biopsies. Focal interstitial lymphocytic infiltration (ILI) was found in 9 (36%) obstructive biopsies whereas in non-obstructive patterns 32 (38.1%) had focal and 27 (32.1%) had wide spread ILI. In NOA late spermatids were present in 32 (38.1%) biopsies. Finding late spermatids was significantly highest when the infiltrate was focal and significantly lowest when the infiltrate was wide spread. Conclusion: In NOA, the possibility of finding an area of complete focal spermatogenesis in the open testicular biopsy is significantly highest when there is a focal type of ILI, but the possibility is significantly lowest when the infiltrate is widespread. This may guide the clinical testicular sperm extraction (TESE) procedure.

Seipin deficiency increases chromocenter fragmentation and disrupts acrosome formation leading to male infertility.

The Berardinelli–Seip congenital lipodystrophy type 2 (Bscl2, seipin) gene is involved in adipogenesis. Bscl2−/− males were infertile but had normal mating behavior. Both Bscl2−/− cauda epididymis sperm count and sperm motility were ~20 × less than control. Bscl2−/− seminiferous tubules had relatively normal presence of spermatogonia and spermatocytes but had reduced spermatids and sperm. Spatiotemporal expression analyses in Bscl2+/+ testes demonstrated prominent Bscl2 transcriptional activity in spermatocytes with a plateau reached around postnatal day 28. Seipin protein localization was most abundant in postmeiotic spermatids, suggesting translational repression of Bscl2 mRNA in spermatocytes. In situ end-labeling plus detected increased spermatid apoptosis in Bscl2−/− testis and annexin V detected increased percentage of positive Bscl2−/− round spermatids compared with control. Immunofluorescence of marker proteins synaptonemal complex proteins 3 and 1 (SYCP3 and SYCP1), and H3K9me3 (histone H3 trimethylated at lysine 9) in germ cell spreads detected normal meiotic chromosome pairing and homologous chromosome synapsis in Bscl2−/− spermatocytes, but significantly increased percentages of round spermatids with chromocenter fragmentation and late spermatids and sperm with chromatin vacuoles, indicating defective chromatin condensation in Bscl2−/− spermatids. Bscl2−/− late spermatids were disorganized within the seminiferous epithelium, despite normal appearance of Sertoli cells detected by vimentin immunofluorescence. Peanut agglutinin staining revealed various abnormalities of acrosomes in Bscl2−/− late spermatids, including the absence, irregular-shaped, and fragmented acrosomes, indicating defective acrosome formation in Bscl2−/− late spermatids, which may affect late spermatid orientation in the seminiferous epithelium. Mitotracker strongly stained the midpiece of control sperm but only very weakly labeled the midpiece of Bscl2−/− sperm, indicating defective mitochondrial activity that most likely contributed to reduced Bscl2−/− sperm motility. These data demonstrate novel roles of seipin in spermatid chromatin integrity, acrosome formation, and mitochondrial activity. Increased spermatid apoptosis, increased chromocenter fragmentation, defective chromatin condensation, abnormal acrosome formation, and defective mitochondrial activity contributed to decreased sperm production and defective sperm that resulted in Bscl2−/− male infertility.

Ultrastructural observations on spermiogenesis in the peanut worm, Phascolosoma esculenta (Sipuncula: Phascolosomatidea)

Ultrastructural characteristics of spermiogenesis in the peanut worm, Phascolosoma esculenta (Phascolosomatidea), were observed by transmission electron microscopy (TEM). The spermiogenesis principally occurs in the coelom and can be divided into three stages (the early, middle and late stages) based on the chromatin morphology, acrosome development, spermatozoon midpiece and flagellum formation. Spermatids within a given spermatid mass develop synchronously. With the spermiogenesis proceeding, the spermatid masses become loosely structured, and later, adjacent spermatids are interconnected at one extremity of the cells. Gradually, condensation of the chromatin accelerates and is almost completed in late spermiogenesis, leaving the late spermatids with highly condensed homogeneous chromatin. In the spermatid head, the conical acrosome is generally composed of an acrosomal vesicle which is formed by the coalescence of small proacrosomal vesicles within the cytoplasm, a subacrosomal space that situates between the acrosomal vesicle and nucleus, and an acrosomal rod which develops from a bunch of filamentous material within the subacrosomal space. Certain mitochondria move posteriorly towards the nucleus, thus constituting the spermatozoon midpiece. The flagellum, originated from the distal centriole, appears in the early spermiogenesis. Ultimately, mature spermatidium dissociates into numerous spermatozoa, which are subsequently released as a single cell from the coelom into the nephridia. The spermatozoon has a prominent head, containing an acrosome and nucleus, a short midpiece and a slender tail. When compared with other sipunculans or invertebrates with external fertilization, the spermiogenesis of P. esculenta, presumably, is closely associated with its biological adaptations for the reproductive strategy.

Characterization of BRD4 during mammalian postmeiotic sperm development.

During spermiogenesis, the postmeiotic phase of mammalian spermatogenesis, transcription is progressively repressed as nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyperacetylation and replacement of most histones with protamines. Although BRDT functions in transcription and histone removal in spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of spermatogenic cells revealed BRD4 in a ring around the nuclei of spermatids containing hyperacetylated histones. The ring lies directly adjacent to the acroplaxome, the cytoskeletal base of the acrosome, previously linked to chromatin reorganization. The BRD4 ring does not form in acrosomal mutant mice. Chromatin immunoprecipitation followed by sequencing in spermatids revealed enrichment of BRD4 and acetylated histones at the promoters of active genes. BRD4 and BRDT show distinct and synergistic binding patterns, with a pronounced enrichment of BRD4 at spermatogenesis-specific genes. Direct association of BRD4 with acetylated H4 decreases in late spermatids as acetylated histones are removed from the condensing nucleus in a wave following the progressing acrosome. These data provide evidence of a prominent transcriptional role for BRD4 and suggest a possible removal mechanism for chromatin components from the genome via the progressing acrosome as transcription is repressed and chromatin is compacted during spermiogenesis.

Light and electron microscopic study on the effect of antischizophrenic drugs on the structure of seminiferous tubules of adult male albino rats.

Sexual dysfunction and infertility are symptoms which have been rarely studied in patients treated with antischizophrenic drugs, aripiprazole and olanzapine, for long period. This work aimed to investigate the effects of aripiprazole and olanzapine on the structure of seminiferous tubules of rats at both light microscopic and ultrastructural levels. Sixty adult male rats were divided into 3 groups (n = 20): control group (Group I) and two experimental ones (II and III). Rats in Group II received 2 mg/kg/day aripiprazole while rats in Group III received 0.5 mg/kg/day olanzapine for 14 weeks. Thereafter, testis were removed and processed for both light and electron microscopic study. Qualitative morphological analyses and histomorphometric measurements of seminiferous tubules were performed. Rats in Group II showed reduction of testicular weight, seminiferous tubules' diameter, epithelial height, spermatogenic count, spermatogenic index and spermatogenic score whereas Sertoli cells count was increased. Olanzapine-treated rats also showed epithelial desquamation, separation and apoptotic changes of germ cells. Sertoli cells showed vacuolization, dilatation of smooth endoplasmic reticulum and accumulation of lipid droplets. Abnormality in the shape and structure of late spermatids and presence of giant cells were also demonstrated. Aripiprazole induced less adverse histological changes in rat testis than olanzapine. Olanzapine followed by aripiprazole had adverse histological effects on the structure of the seminiferous tubules, which may affect spermatogenesis.

Zygotic chromosomal structural aberrations after paternal drug treatment.

In recent years, the field of male-mediated reproductive toxicology has received growing attention. It is now well-established that many drugs, chemicals, and environmental factors can harm male germ cells by inducing DNA damage. Male germ cells have extensive repair mechanisms that allow detection and repair of damaged DNA during the early phases of spermatogenesis. However, during the later phase of spermiogenesis, when the haploid spermatids undergo chromatin condensation and become transcriptionally quiescent, their ability to repair damaged DNA is lost.12 It is also thought that the highly compacted chromatin of the sperm can protect DNA against damage.3 Therefore, it is expected that late spermatids will be most susceptible to DNA damaging agents. Unrepaired or misrepaired damage in the germ cells leads to the generation of spermatozoa with DNA damage that can be transmitted to the next generation. Fortunately, the maternal DNA repair machinery is capable of recognizing and repairing, at least to some degree, damaged paternal DNA after fertilization in the zygote. Therefore, the efficiency of the maternal repair machinery will greatly influence the risk of transmitting paternal DNA damage to offspring.4

Spermatogenesis and spermatozoa ultrastructure of two Dipolydora species (Annelida: Spionidae) from the Sea of Japan

Spermatogenesis and the structure of the spermatozoa of two spionid polychaetes Dipolydora bidentata and Dipolydora carunculata are described by light and transmission electron microscopy. Both species are gonochoristic borers in shells of various molluscs. Proliferation of spermatogonia occurs in paired testes regularly arranged in fertile segments, and the rest of spermatogenesis occurs in the coelomic cavity. Early spermatogenesis occurs quite similarly in the two species but results in formation of tetrads of interconnected spermatids in D. bidentata and octads of spermatids in D. carunculata. Three consecutive stages of spermiogenesis are recognized according to the condensation of chromatin in nucleus: (1) early spermatids with heterogeneous, partly clumped chromatin, (2) middle spermatids with homogeneous, coarsely granular chromatin, and (3) late spermatids with homogeneous fibrillar chromatin. Moreover, late stage of spermatids is further classified into two stages, I and II, according to the position of the acrosome and shape of the nucleus. In late spermatids I, the acrosome is situated in the anterior invagination of the funnel-shaped to oval nucleus, whereas in late spermatids II the acrosome is situated on top of the elongated nucleus. Ultrastructural composition of cells at each stage of spermatogenesis is described and illustrated. The possible process of morphogenesis of organelles during spermato- and spermiogenesis is reconstructed for both species. The proacrosomal vesicle first appears in early spermatids near the Golgi complex and then migrates anteriorly; in the middle spermatids, the acrosome comes to lie in a deep anterior nuclear fossa. In late spermatids I, this fossa evaginates and a posterior fossa develops in the nucleus housing basal body and the anterior part of the axoneme. In late spermatids II, the mitochondria elongate and probably reduce in number due to fusion of some of them. The mature spermatozoa in both species are introsperm with the conical acrosome, subacrosomal plate, long nucleus with short posterior fossa, long midpiece with elongated mitochondria, and long flagellum with 9×2+2 organization of microtubules. Numerous flat rounded platelets with putative glycogen are present throughout most part of the nucleus and the midpiece. The process of spermatogenesis in D. bidentata and D. carunculata is similar to that in other Dipolydora, Polydora and Pseudopolydora species. Spermatozoa in these polydorin spionids have similar composition and differ mainly in size of the nucleus and the midpiece. Elongated spermatozoa are adapted for transfer in spermatophores and an internal fertilization which is characteristic for brooding species. Diversely modified spermatozoa among spionids may be signs of the diversity of fertilization biology within the Spionidae. The exact places where fertilization occurs in brooding spionids however remains unknown.

Mechanisms of translational repression of the Smcp mRNA in round spermatids

The protamine 1 (Prm1) and sperm mitochondria-associated, cysteine-rich protein (Smcp) mRNAs exemplify a widespread pattern of mRNA-specific regulation of mRNA translation in post-meiotic spermatogenic cells, spermatids. Both mRNAs are transcribed and initially stored in free-mRNPs in early spermatids, and translated on polysomes in late spermatids. In this study, we demonstrate that the 5' and 3'-UTRs and the 3' terminus of the Smcp 3'-UTR are required for normal repression of the Smcp mRNA in transgenic mice. RNA affinity chromatography and mass spectrometry sequencing identified Y-box protein 2 (YBX2/MSY2) as the major protein that interacts with the 3' terminus of the Smcp 3'-UTR and a Y-box recognition sequence, GCCACCU, in the translation control element that is necessary for Prm1 mRNA repression. Depletion of YBX2 in Ybx2-null mice prematurely activates Prm1 and Smcp mRNA translation in early spermatids. Fluorescent in situ hybridization reveals that the Smcp intron, the Smcp mRNA, and both Smcp-Gfp transgenic mRNAs are strongly concentrated in the chromatoid body, and that theYbx2-null mutation does not eliminate the Smcp mRNA from the chromatoid body. This and previous findings suggest that the Smcp pre-mRNA is spliced and associates with YBX2 in the chromatoid body, and that repressed free-mRNPs are stored in the general cytoplasm. As YBX2 is the predominant protein in testis free-mRNPs, it likely represses many mRNAs in early spermatids. The mechanisms by which YBX2 represses the Smcp and Prm1 mRNAs are relevant to reproductive medicine because mutations in the human YBX2 gene correlate with abnormal protamine expression and male infertility.

Increasing testicular temperature by exposure to elevated ambient temperatures restores spermatogenesis in adult Utp14b (jsd) mutant (jsd) mice.

Because mutations in the human UTP14C gene are associated with male infertility, we sought to develop a method for fertility restoration in azoospermic mice with a mutation in the orthologous Utp14b(jsd) (jsd) gene that have spermatogonial arrest. The method is based on our observation that elevation of testicular temperatures restores spermatogonial differentiation in jsd mutant mice. To non-surgically raise intrascrotal temperatures we placed these mice in incubators at different elevated ambient temperatures. Exposure of jsd/jsd mice to ambient temperatures of 34.5 °C or 35.5 °C for 24 days increased the proportion of tubules with spermatocytes from 0% in untreated controls to over 80%. As those higher temperatures interfere with spermatid differentiation, the mice were then transferred to incubators at 32-32.5 °C for the next 24 days. These environments allowed differentiation to progress, resulting in up to 42% of tubules having late spermatids and about half of the mutant mice having spermatozoa in testicular suspensions. When these spermatozoa were used in intracytoplasmic sperm injection, all gave rise to viable healthy offspring with normal weight gain and fertility. The successful restoration of fertility in Utp14b mutant mice suggests that transient testicular warming might also be useful for spermatogenesis recovery in infertile men with UTP14C gene mutations.

Functional Analysis of KIF3A and KIF3B during Spermiogenesis of Chinese Mitten Crab Eriocheir sinensis.

BackgroundSpermatogenesis represents the transformation process at the level of cellular development. KIF3A and KIF3B are believed to play some roles in the assembly and maintenance of flagella, intracellular transport of materials including organelles and proteins, and other unknown functions during this process. During spermatogenesis in Eriocheir sinensis, if the sperm shaping machinery is dependent on KIF3A and KIF3B remains unknown.Methodology/Principal FindingsThe cDNA of KIF3A and KIF3B were obtained by designing degenerate primers, 3′RACE, and 5′RACE. We detected the genetic presence of kif3a and kif3b in the heart, muscle, liver, gill, and testis of E. sinensis through RT-PCR. By western blot analysis, the protein presence of KIF3A and KIF3B in heart, muscle, gill, and testis reflected the content in protein level. Using in situ hybridization and immunofluorescence, we could track the dynamic location of KIF3A and KIF3B during different developmental phases of sperm. KIF3A and KIF3B were found surrounding the nucleus in early spermatids. In intermediate spermatids, these proteins expressed at high levels around the nucleus and extended to the final phase. During the nuclear shaping period, KIF3A and KIF3B reached their maximum in the late spermatids and were located around the nucleus and concentrated in the acrosome to some extent.Conclusions/SignificanceOur results revealed that KIF3A and KIF3B were involved in the nuclear and cellular morphogenesis at the levels of mRNA and protein. These proteins can potentially facilitate the intracellular transport of organelles, proteins, and other cargoes. The results represent the functions of KIF3A and KIF3B in the spermatogenesis of Crustacea and clarify phylogenetic relationships among the Decapoda.

A multicopy Y-chromosomal SGNH hydrolase gene expressed in the testis of the platyfish has been captured and mobilized by a Helitron transposon

BackgroundTeleost fish present a high diversity of sex determination systems, with possible frequent evolutionary turnover of sex chromosomes and sex-determining genes. In order to identify genes involved in male sex determination and differentiation in the platyfish Xiphophorus maculatus, bacterial artificial chromosome contigs from the sex-determining region differentiating the Y from the X chromosome have been assembled and analyzed.ResultsA novel three-copy gene called teximY (for testis-expressed in Xiphophorus maculatus on the Y) was identified on the Y but not on the X chromosome. A highly related sequence called texim1, probably at the origin of the Y-linked genes, as well as three more divergent texim genes were detected in (pseudo)autosomal regions of the platyfish genome. Texim genes, for which no functional data are available so far in any organism, encode predicted esterases/lipases with a SGNH hydrolase domain. Texim proteins are related to proteins from very different origins, including proteins encoded by animal CR1 retrotransposons, animal platelet-activating factor acetylhydrolases (PAFah) and bacterial hydrolases. Texim gene distribution is patchy in animals. Texim sequences were detected in several fish species including killifish, medaka, pufferfish, sea bass, cod and gar, but not in zebrafish. Texim-like genes are also present in Oikopleura (urochordate), Amphioxus (cephalochordate) and sea urchin (echinoderm) but absent from mammals and other tetrapods. Interestingly, texim genes are associated with a Helitron transposon in different fish species but not in urochordates, cephalochordates and echinoderms, suggesting capture and mobilization of an ancestral texim gene in the bony fish lineage. RT-qPCR analyses showed that Y-linked teximY genes are preferentially expressed in testis, with expression at late stages of spermatogenesis (late spermatids and spermatozeugmata).ConclusionsThese observations suggest either that TeximY proteins play a role in Helitron transposition in the male germ line in fish, or that texim genes are spermatogenesis genes mobilized and spread by transposable elements in fish genomes.

PRKRA Localizes to Nuage Structures and the Ectoplasmic Specialization and Tubulobulbar Complexes in Rat and Mouse Testis

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including PRKRA, TRBP, and Dicer. RISC localizes to P-bodies. The nuage of the spermatogenic cells has function similar to the P-bodies. We study whether PRKRA localizes to nuage of spermatogenic cells of rat and mouse. PRKRA localized to four types of nuage structures, including aggregates of 60–90 nm particles, irregularly-shaped perinuclear granules, and intermitochondrial cement of pachytene spermatocytes, and chromatoid bodies of round spermatids. In addition, PRKRA is associated with dense material surrounding tubulobulbar complexes and with the ectoplasmic specialization. The results suggest that PRKRA functions in the nuage as an element of RNA silencing system and plays unknown role in the ectoplasmic specialization and at the tubulobulbar complexes of Sertoli cells attaching the head of late spermatids.

DGCR8 Localizes to the Nucleus as well as Cytoplasmic Structures in Mammalian Spermatogenic Cells and Epididymal Sperm

The localization of DGCR8 in spermatogenic cells and sperm from rat and mouse was studied by immunofluorescence and immunoelectron microscopy. Spermatogenic cells from these species yielded similar DGCR8 localization pattern. Immunofluorescence microscopy results showed that DGCR8 localized to both the cytoplasm and nucleus. In the cytoplasm, diffuse cytosolic and discrete granular staining was observed. Dual staining showed that DGCR8 colocalized to the granules with MAEL (a nuage marker). In the nucleus of spermatocytes, both the nucleoli and nucleoplasm were stained, whereas in the nucleus of early spermatids small spots were stained. In late spermatids, DGCR8 localized to the tip of their head and to small granules (neck granules) of the neck cytoplasm. The neck granules were also observed in the neck of epididymal sperm. Immunoelectron microscopy results showed that DGCR8 localized to nuage structures. Moreover, DGCR8 localized to nonnuage structures in late spermatids. DGCR8 also localized to the nucleolus and euchromatin in spermatocytes and round spermatids and to small granules in the nucleus of late spermatids. The results suggest that in spermatogenic cells DGCR8 localizes not only to the nuclei but also to the cytoplasmic structures such as nuage and nonnuage structures. Furthermore, DGCR8 seems to be imported into the egg with neck granules in sperm during fertilization.

Development of spermatogenic cells and sperm quality after administration of pegagan (Centella asiatica) extract

The purpose of this study was to determine the development of spermatogenic cells and sperm quality after administration extracts of pegagan (Centella asiatica) in various doses and duration of administration. The research was carried out with complete randomized design (CRD), consist of 16 combinations of dose and duration of treatment. Parameters measured consist of population of spermatogenic cells (spermatogonia, primary spermatocytes, late spermatids) and sperm quality (concentration, motility, abnormality). The data were processed using analysis of variance (ANOVA), and differences between treatments followed by Duncan test. The results show that both the dose and duration have very significantly (p < 0.01) affect on decreasing of late spermatid population, sperm motility and concentration, but not for population of spermatogonia, primary spermatocytes and sperm abnormalities. The decrease of population and quality may due to antifertility effect of pegagan, eventhough still in the normal range. It is concluded that spermatogenic cells development and sperm quality reduce after administration of pegagan extract, although infertility is not yet found up to the dose of 450 mg/kg BW for 49 days duration of administration. Key Words : Spermatogenic, Sperm Quality, Pegagan Extract

DDX6 localizes to nuage structures and the annulus of mammalian spermatogenic cells

The localization of DEAD (Asp-Glu-Ala-Asp) box helicase 6 (DDX6) in spermatogenic cells from the mouse, rat, and guinea pig was studied by immunofluorescence (IF) and immunoelectron microscopy (IEM). Spermatogenic cells from these species yielded similar DDX6 localization pattern. IF microscopy results showed that DDX6 localizes to both the nucleus and cytoplasm. In the cytoplasm of spermatogenic cells, diffuse cytosolic and discrete granular staining was observed, with the staining pattern changing during cell differentiation. IEM revealed that DDX6 localized to the five different types of nuage structures and non-nuage structures, including small granule aggregate and late spermatid annuli. Nuclear labeling was strongest in leptotene and zygotene spermatocytes and moderately strong in the nuclear pocket of late spermatids. DDX6 also localized to the surface of outer dense fibers, which comprise of flagella. The results show that DDX6 is present in nuage and non-nuage structures as well as nuclei, suggesting that DDX6 has diverse functions in spermatogenic cells.

Molecular characterization and expression analysis of a KIFC1-like kinesin gene in the testis of Eumeces chinensis.

The member of the kinesin-14 subfamily, KIFC1, is a carboxyl-terminal motor protein that plays an important role in the elongation of nucleus and acrosome biogenesis during the spermiogenesis of mammals. Here, we had cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed ec-KIFC1) from the total RNA of the testis of the reptile Eumeces chinensis. The full-length sequence was 2,339bp that contained a 216bp 5'-untranslated region (5'UTR), a 194bp 3'-untranslated region (3'UTR) and a 1,929bp open reading frame that encoded a special protein of 643 amino acids (aa). The calculated molecular weight of the putative ec-KIFC1 was 71kDa and its estimated isoelectric point was 9.47. The putative ec-KIFC1 protein owns a tail domain from 1 to 116aa, a stalk domain from 117 to 291aa and a conserved carboxyl motor domain from 292 to 642aa. Protein alignment demonstrated that ec-KIFC1 had 45.6, 42.8, 44.6, 36.9, 43.7, 46.4, 45.1, 55.6 and 49.8% identity with its homologues in Mus musculus, Salmo salar, Danio rerio, Eriocheir sinensis, Rattus norvegicus, Homo sapiens, Bos taurus, Gallus gallus and Xenopus laevis, respectively. Tissue expression analysis showed the presence of ovary, heart, liver, intestine, oviduct, testis and muscle. The phylogenetic tree revealed that ec-KIFC1 was more closely related to vertebrate KIFC1 than to invertebrate KIFC1. In situ hybridization showed that the ec-KIFC1 mRNA was localized in the periphery of the nuclear membrane and the center of the nucleus in early spermatids. In mid spermatids, the ec-KIFC1 had abundant expression in the center of nucleus, and was expressed in the tail and the anterior part of spermatids. In the late spermatid, the nucleus gradually became elongated, and the ec-KIFC1 mRNA signal was still centralized in the nucleus. In mature spermatids, the signal of the ec-KIFC1 gradually became weak, and was mainly located at the tail of spermatids. Therefore, the ec-KIFC1 probably plays a critical role in the spermatogenesis of E. chinensis.

Sperm characteristics as additional evidence of close relationship between Lebiasina and Piabucina (Characiformes: Lebiasinidae: Lebiasininae)

Spermiogenesis and spermatozoa of representatives of the genera Lebiasina and Piabucinaare described. Spermiogenesis is quite similar among all analyzed species of these genera and is identified as Type II. In this type of spermiogenesis, the flagellum of earliest spermatids lies lateral to the nucleus. A slight movement of the nucleus towards the centriolar complex is observed. In late spermatids, the nucleus slightly elongates towards the flagellum. The formation of two striated rootlets apparently occurs in early/intermediate spermatids. The spermatozoa of species of Lebiasinaand Piabucina share (1) a drop-shaped slightly elongated nucleus that is laterally positioned relative to the flagellum; (2) a superolateral centriolar complex; (3) two striated rootlets; (4) a basolateral midpiece; (5) oblong mitochondria and (6) a few spherical vesicles. The spermatic characteristics of Lebiasinaand Piabucinaindicate that spermiogenesis is a homologous process among all species of both genera examined to date and can be considered, along with the spermatozoa morphology, additional evidence indicating a close relationship between Lebiasina and Piabucina.

A network of spectrin and plectin surrounds the actin cuffs of apical tubulobulbar complexes in the rat

Tubulobulbar complexes (TBCs) are actin-related endocytic structures that internalize intercellular junctions in the seminiferous epithelium. The structures consist of elongate tubular projections of the attached plasma membranes of two adjacent cells that project into Sertoli cells. This double membrane core is cuffed by a dentritic actin network and is capped at its end by a clathrin-coated pit. Here we explore the possibility that elements of the spectrin cytoskeleton are associated with clusters of tubulobulbar complexes that develop at adhesion junctions between late spermatids and Sertoli cells at the apex of the epithelium, and extend what is known about the distribution of plectin at the sites.Cryo-sections of perfusion-fixed testes and apical processes of Sertoli cells mechanically dissociated from perfusion-fixed testes were probed for spectrin, EPB41, and actin and analyzed using conventional fluorescence microscopy and confocal microscopy. Data sets from confocal microscopy were analyzed further in three-dimensional reconstructions using computer software. Additional apical Sertoli cell processes were probed for plectin and analyzed using conventional fluorescence microscopy. Antibodies generated against elements of the spectrin cytoskeleton react with material around and between the actin cuffs of tubulobulbar complexes, but appear excluded from the actin cuffs themselves. A similar staining pattern occurs with a probe for plectin. Immunoelectron microscopy confirmed the staining patterns observed by fluourescence microscopy. Based on our results, we suggest that a network of spectrin and plectin forms a scaffold around tubulobulbar complexes that may provide support for the actin network that cuffs each complex and also link adjacent complexes together.

Breast cancer resistance protein (Bcrp) and the testis—an unexpected turn of events

Breast cancer resistance protein (Bcrp) is an ATP-dependent efflux drug transporter. It has a diverse spectrum of hydrophilic and hydrophobic substrates ranging from anticancer, antiviral and antihypertensive drugs, to organic anions, antibiotics, phytoestrogens (e.g., genistein, daidzein, coumestrol), xenoestrogens and steroids (e.g., dehydroepiandrosterone sulfate). Bcrp is an integral membrane protein in cancer and normal cells within multiple organs (e.g., brain, placenta, intestine and testis) that maintains cellular homeostasis by extruding drugs and harmful substances from the inside of cells. In the brain, Bcrp is a major component of the blood-brain barrier located on endothelial cells near tight junctions (TJs). However, Bcrp is absent at the Sertoli cell blood-testis barrier (BTB); instead, it is localized almost exclusively to the endothelial TJ in microvessels in the interstitium and the peritubular myoid cells in the tunica propria. Recent studies have shown that Bcrp is also expressed stage specifically and spatiotemporally by Sertoli and germ cells in the seminiferous epithelium of rat testes, limited only to a testis-specific cell adhesion ultrastructure known as the apical ectoplasmic specialisation (ES) in stage VI-early VIII tubules. These findings suggest that Bcrp is equipped by late spermatids and Sertoli cells to protect late-stage spermatids completing spermiogenesis. Furthermore, Bcrp was found to be associated with F (filamentous)-actin and several actin regulatory proteins at the apical ES and might be involved in the organisation of actin filaments at the apical ES in stage VII-VIII tubules. These findings will be carefully evaluated in this brief review.