Laura L. Tres

Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures☆

Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20-22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio-immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM-C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process.

The Acroplaxome Is the Docking Site of Golgi-Derived Myosin Va/Rab27a/b- Containing Proacrosomal Vesicles in Wild-Type and Hrb Mutant Mouse Spermatids

Abstract Acrosome biogenesis involves the transport and fusion of Golgi-derived proacrosomal vesicles along the acroplaxome, an F-actin/keratin 5-containing cytoskeletal plate anchored to the spermatid nucleus. A significant issue is whether the acroplaxome develops in acrosomeless mutant mice. Male mice with a Hrb null mutation are infertile and both spermatids and sperm are round-headed and lack an acrosome. Hrb, a protein that contains several NPF motifs (Asn-Pro-Phe) and interacts with proteins with Eps15 homology domains, is regarded as critical for the docking and/or fusion of Golgi-derived proacrosomal vesicles. Here we report that the lack of an acrosome in Hrb mutant spermatids does not prevent the development of the acroplaxome. Yet the acroplaxome in the mutant contains F-actin but is deficient in keratin 5. We also show that the actin-based motor protein myosin Va and its receptor, Rab27a/b, known to be involved in vesicle transport, are present in the Golgi and Golgi-derived proacrosomal vesicles in wild-type and Hrb mutant mouse spermatids. In the Hrb mutant, myosin-Va-bound proacrosome vesicles tether to the acroplaxome, where they flatten and form a flat sac, designated pseudoacrosome. As spermiogenesis advances, round-shaped spermatid nuclei of the mutant display several nuclear protrusions, designated nucleopodes. Nucleopodes are consistently found at the acroplaxome- pseudoacrosome site. Our findings support the interpretation that the acroplaxome provides a focal point for myosin-Va/ Rab27a/b-driven proacrosomal vesicles to accumulate, coalesce, and form an acrosome in wild-type spermatids and a pseudoacrosome in Hrb mutant spermatids. We suggest that nucleopodes develop at a site where a keratin 5-deficient acroplaxome may not withstand tension forces operating during spermatid nuclear shaping.

Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis

We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.

Nucleolar RNA synthesis of meiotic prophase spermatocytes in the human testis

Human meiotic prophase spermatocyte nuclei were studied by electron microscope autoradiography after a 3 hours 3H-uridine labeling pulse, followed by postincubation in non-radioactive medium. In autosomes, 3H-uridine nucleolar labeling reaches a peak during early-middle zygotene prior to the peak labeling of chromosomal RNA species at middle pachytene. Transcription activities of sex chromosomes are inconspicuous when compared with that of autosomes. An increasing condensation of nucleolar-associated chromatin in acrocentric bivalents contributes to the formation of basal knobs in human pachytene spermatocytes. Upon completion of knob formation, nucleolar components segregate and the uptake of 3H-uridine decreases. These findings suggest that the template capability of ribosomal DNA cistrons, located next to the basal knob region, is largely associated with a dispersed state of chromatin whereas increased chromatin condensation is correlated with a restriction of ribosomal RNA transcription.

A protein associated with the manchette during rat spermiogenesis is encoded by a gene of the TBP‐1‐like subfamily with highly conserved ATPase and protease domains

We have used a rat pachytene spermatocyte cDNA expression library to clone TBP‐1 (for tat‐binding protein‐1; designated rat testis TBP‐1 [rtTBP‐1]), a new member of the family of putative ATPases associated with the 26S proteasome complex. The 1.63 kb rtTBP‐1 cDNA encodes a 49 kDa protein with 99% amino acid identity to human TBP‐1 protein. rtTBP‐1 protein contains a heptad repeat of six leucine‐type zipper fingers at the amino terminal end and highly conserved ATPase and DNA/RNA helicase motifs towards the carboxyl terminal region. Chromatofocusing fractionation of rat testis sucrose extracts demonstrates that the encoded product, recognized by an antiserum raised to the first 196 amino acids of human TBP‐1, consists of a protein triplet with a molecular mass range of 52‐48 kDa and acidic pI (5.0–5.9). An identical immunoreactive triplet was detected by immunoblotting in extracts of fractionated pachytene spermatocytes, round spermatids and epididymal sperm. In situ hybridization using digoxigenin‐labeled antisense RNA probes shows a predominant distribution of specific mRNA in the seminiferous epithelial region occupied by elongating spermatids and primary spermatocytes. Indirect immunofluorescence and immunogold electron microscopy studies show that rtTBP‐1 immunoreactive sites colocalize with α‐tubulin‐decorated manchettes of elongating spermatids. In addition, rtTBP‐1 immunoreactivity was detected in fibrillar and granular cytoplasmic bodies typically observed in spermatocytes and spermatids as well as in association with paraaxonemal mitochondria and outer dense fibers of the developing spermatid tail. Results of this study indicate that rtTBP‐1 is a member of the highly evolutionary conserved TBP‐1‐like subfamily of putative ATPases, sharing regions of identity—including ATP‐binding sites—with several subunits of the 26S proteasome, known to be involved in the ATP‐dependent degradation of ubiquitin‐conjugated proteins. Mol. Reprod. Dev. 48:77–89, 1997.

Mouse Round Spermatids Developed In Vitro from Preexisting Spermatocytes Can Produce Normal Offspring by Nuclear Injection into In Vivo-Developed Mature Oocytes

Abstract It has been shown that mature oocytes injected with nuclei from round spermatids collected from mouse testis can generate normal offspring and that round spermatids can develop in vitro. An undetermined issue is whether spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (γ-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy (http://www.sci.ccny.cuny.edu/∼kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins.

Temporal sequence of cell shape changes in cultured rat sertoli cells after experimental elevation of intracellular cAMP.

Abstract The ability of FSH and pharmacological agents to induce changes in the shape of cultured rat Sertoli cells has been studied by using time-lapse phase-contrast microscopy and scanning electron microscopy (SEM). Morphological studies were combined with an immunocytochemical method for the localization of cAMP in Sertoli cells and the results correlated with determinations of protein-bound cAMP in Sertoli cells. A variable number of Sertoli cells were converted from a flat, epithelial-like morphology into a stellate morphology after exposure to FSH, isobutyl-methylxanthine (MIX), dibutyryl cyclic AMP (db-cAMP) and an FSH-MIX mixture. The morphological changes followed a time- and biological agent-dependent alteration and recovery pattern. While a 120 min exposure to FSH induced shape changes in 38% of the cells, MIX, db-cAMP and FSH-MIX effected shape changes in 75 % of cells. The morphological conversion induced by MIX, db-cAMP and FSH-MIX persisted as long as these biological agents were present in the medium, whereas the effects induced by FSH alone were transient. The flat-to-stellate transition was preceded by an increase in intracellular protein-bound cAMP, a form of cyclic nucleotide which may account for cAMP immunoreactivity observed in morphologically responsive and non-responsive Sertoli cells. From these data and from previous experimental findings of androgen-binding protein (ABP) immunoreactivity in the cytoplasm of responsive and non-responsive Sertoli cells, we conclude that a surge of cAMP triggers a still undefined mechanism by which Sertoli cells modify their shape in coincidence with a progressive depletion of cytoplasmic secretory granules.

Cytoskeletal track selection during cargo transport in spermatids is relevant to male fertility

Spermatids generate diverse and unusual actin and microtubule populations during spermiogenesis to fulfill mechanical and cargo transport functions assisted by motor and non-motor proteins. Disruption of cargo transport may lead to teratozoospermia and consequent male infertility. How motor and non-motor proteins utilize the cytoskeleton to transport cargos during sperm development is not clear. Filamentous actin (F-actin) and the associated motor protein myosin Va participate in the transport of Golgi-derived proacrosomal vesicles to the acrosome and along the manchette. The acrosome is stabilized by the acroplaxome, a cytoskeletal plate anchored to the nuclear envelope. The acroplaxome plate harbors F-actin and actin-like proteins as well as several other proteins, including keratin 5/Sak57, Ran GTPase, Hook1, dynactin p150Glued, cenexin-derived ODF2, testis-expressed profilin-3 and profilin-4, testis-expressed Fer tyrosine kinase (FerT), members of the ubiquitin-proteasome system, and cortactin. Spermatids express transcripts encoding the non-spliced form of cortactin, a F-actin-regulatory protein. Tyrosine phosphorylated cortactin and FerT coexist in the acrosome-acroplaxome complex. Hook1 and p150Glued, known to participate in vesicle cargo transport, are sequentially seen from the acroplaxome to the manchette to the head-tail coupling apparatus (HTCA). The golgin Golgi-microtubule associated protein GMAP210 resides in the cis-Golgi whereas the intraflagellar protein IFT88 localizes in the trans-Golgi network. Like Hook1 and p150Glued, GMAP210 and IFT88 colocalize at the cytosolic side of proacrosomal vesicles and, following vesicle fusion, become part of the outer and inner acrosomal membranes before relocating to the acroplaxome, manchette, and HTCA. A hallmark of the manchette and axoneme is microtubule heterogeneity, determined by the abundance of acetylated, tysosinated, and glutamylated tubulin isoforms produced by post-translational modifications. We postulate that the construction of the male gamete requires microtubule and F-actin tracks and specific molecular motors and associated non-motor proteins for the directional positioning of vesicular and non-vesicular cargos at specific intracellular sites.

Structural and biochemical features of fractionated spermatid manchettes and sperm axonemes of the Azh/Azh mutant mouse

The tubulin‐containing axoneme and manchette develop consecutively during mammalian spermiogenesis. The nature of their molecular components and developmental sequence are not completely known. The azh/azh (for abnormal sperm headshape) mouse mutant is an ideal model for analyzing tubulin isotypes and microtubule‐associated proteins of the manchette and axoneme in light of a potential role of the manchette in the shaping of the sperm head and formation of the tail. We have searched for possible differences in tubulin isotype variants in fractionated manchettes and axonemes of wildtype and azh/azh mutant mice using isotypespecific tubulin antibodies as immunoprobes. Manchettes from wild‐type and azh/azh mutant mouse spermatids were fractionated from spermatogenic stage‐specific seminiferous tubules and axonemes were isolated from epididymal sperm. We have found that: (1) Fractionated manchettes of azh/azh mutants are longer than in wild‐type mice; (2) Manchette and sperm tail axonemes display a remarkable variety of posttranslationally modified tubulins (acetylated, glutamylated, tyrosinated, α‐3/7 tubulins). Acetylated tubulin was more abundant in manchette than in axonemes; (3) An acidic 62 kDa protein was identified as the main component of the perinuclear ring of the manchette in wild‐type and azh/azh mice; (4) Bending and looping of the mid piece of the tail of azh/azh sperm, accompanied by a dislocation of the connecting piece from head attachment sites, were visualized by phase‐contrast, immunofluorescence and transmission electron microscopy in about 35% of spermatids/sperm; and (5) A lasso‐like tail configuration was predominant in epididymal sperm of azh/azh mutants. We speculate that spermatid and sperm tail abnormalities in the azh/azh mutant could reflect structural and/or assembly deficiencies of peri‐axonemal proteins responsible for maintaining a stiffened tail during spermiogenesis and sperm maturation. Mol. Reprod. Dev. 52:434–444, 1999

GMAP210 and IFT88 are present in the spermatid golgi apparatus and participate in the development of the acrosome–acroplaxome complex, head–tail coupling apparatus and tail

We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of spermatids and the participation of these two proteins in the development of the acrosome–acroplaxome complex, the head–tail coupling apparatus (HTCA) and the spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis‐Golgi, whereas IFT88 prevails in the trans‐Golgi network. Both proteins colocalize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome–acroplaxome region of the sperm head, whereas GMAP210 is no longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail‐less. GMAP210 is visualized in the Ift88 mutant during acrosome–acroplaxome biogenesis. However, GMAP210–stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the spermatid Golgi and microtubules with Brefeldin‐A and nocodazole blocks the progression of GMAP210‐ and IFT88‐stained proacrosomal vesicles to the acrosome–acroplaxome complex but F‐actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of spermatids, that the microtubule‐associated golgin GMAP210 and IFT88 participate in acrosome, HTCA, and tail biogenesis, and that defective intramanchette transport of cargos disrupts spermatid tail development. Developmental Dynamics 240:723–736, 2011.