Abraham L. Kierszenbaum

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.

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.

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.

Rat hd Mutation Reveals an Essential Role of Centrobin in Spermatid Head Shaping and Assembly of the Head-Tail Coupling Apparatus

The hypodactylous (hd) locus impairs limb development and spermatogenesis, leading to male infertility in rats. We show that the hd mutation is caused by an insertion of an endogenous retrovirus into intron 10 of the Cntrob gene. The retroviral insertion in hd mutant rats disrupts the normal splicing of Cntrob transcripts and results in the expression of a truncated protein. During the final phase of spermiogenesis, centrobin localizes to the manchette, centrosome, and the marginal ring of the spermatid acroplaxome, where it interacts with keratin 5-containing intermediate filaments. Mutant spermatids show a defective acroplaxome marginal ring and separation of the centrosome from its normal attachment site of the nucleus. This separation correlates with a disruption of head-tail coupling apparatus, leading to spermatid decapitation during the final step of spermiogenesis and the absence of sperm in the epididymis. Cntrob may represent a novel candidate gene for presently unexplained hereditary forms of teratozoospermia and the “easily decapitated sperm syndrome” in humans.

THE SECRETION OF ANDROGEN‐BINDING PROTEIN AND OTHER PROTEINS BY RAT SERTOLI CELLS IN CULTURE: A STRUCTURAL AND ELECTROPHORETIC STUDY*

In the testis of the sexually mature animal, Sertoli cells maintain structural and functional relationships with germinal cells. Sertoli cells are nonproliferative in the mature animal and maintain a transient association with germinal cells. It is believed that biological signals generated by the functionally cycling Sertoli cells 1. ’? enable the spermatogenic process to reach completion. However, the functional nature of the Sertoli cell-germinal cell relationship is poorly understood. Therefore, it seems relevant to increase our understanding of Sertoli cell physiology as a step towards the study of germinal cell differentiation into spermatozoa. A convenient approach for the assessment of Sertoli cell function is the use of a cell culture system in which physiological activities can be correlated with events occurring in the seminiferous epithelium. Using primary cultures of rat Sertoli cells, it was shown that F S H rcgulates the synthesis and secretion of androgen-binding protein (ABP) ;’ {. a biological marker of Sertoli cells. In the intact tcstis, ABP is released primarily into the seminiferous tubular lumen. transported into the epididymis via testicular fluid and concentrated in the caput epididymis.’ However, the ultimate question is the effect of FSH on Sertoli cell secretory proteins resulting in the regulation of spermatogenesis. In this paper we review the effects of FSH on cultured Sertoli cells isolated from 20to 22-day old rats with spccial reference to thc immunocytochemical localization of ABP and the temporal sequence of Sertoli cell shape changes,: which coincide with a progressive depletion of ABP immunoreactive cytoplasmic granules. In addition, we address ourselves to the question of FSH-regulated secretory proteins other than ABP that appear in the medium generated by cultured Sertoli cells. We have used two-dimensional polyacrylamide gel electrophoresis to study the protein secretory activity of Sertoli cells from 10and 20-day old rats incubated in a serum-free, hormone-supplemented medium. We find that FSH induces the accumulation in the tissue culture medium of several I”Slniethionine-labeled polypeptides that were not detected in the medium of cultured Sertoli cells not stimulated by FSH.

Antigenic homology between rat sperm tail polypeptides and Sertoli cell secretory proteins

AbstractA high performance liquid chromatographic procedure has been used for the purification of rat Sertoli cell secretory protein S70 and S45-535 heterodimeric protein to determine their role during spermatogenesis. These two proteins display binding affinity for each other and appear antigenically related. We have observed that:1.S70 and S45-S35 heterodimeric protein coelute during purification,2.polyclonal antiserum raised against protein S70 recognizes common antigenic determinants in polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein, and3.a monoclonal antibody that recognizes polypeptide S35 but does not crossreact with either protein S70 or polypeptide S45, immunoprecipitates the S70/S45-S35 heterodimeric protein complex.nIn immunofluorescent experiments, antisera raised against protein S70 and polypeptide components of S45-S35 heterodimeric protein immunoreact with two major sperm intracellular structures: the acrosome and periaxonemal outer dense fibers of sperm tail. Immunoreactivity was not detected on the sperm plasma membrane surface of unfixed, living sperm. Outer dense fibers extracted from sperm tails by a combined treatment with cetylthrimethylammonium bromide and 2-mercaptoethanol, yielded a characteristic polypeptide pattern. In immunoblotting experiments, sperm tail polypeptides were recognized by polyclonal antisera raised against Sertoli cell secretory proteins. We conclude that Sertoli cell secretory proteins S70 and S45-S35 heterodimeric protein are antigenically related to each other and to keratin-like polypeptides from sperm tail.

Isolation and culture of rat seminal vesicle epithelial cells: The use of the secretory protein SVS IV as a functional probe

A method for the isolation and culture of seminal vesicle epithelial cells obtained from control and androgen-primed sexually-immature, uncastrated rats is described. This method allows the establishment of monolayer cultures from aggregates of seminal vesicle epithelial cells isolated after trypsin and collagenase digestion. Phase contrast and transmission electron microscopic methods demonstrate that cell aggregates, after attaching to the substrate, establish within 48 h a colony-like, epithelial-like growth pattern. Immunofluorescent localization studies of SVS IV, an androgen-dependent secretory protein purified from rat seminal vesicle secretion, show that cultured seminal vesicle epithelial cells are immunoreactive. An electrophoretic analysis of [35S]methionine-labeled secretory proteins immunoprecipitated with rabbit anti-SVS IV serum demonstrate that, whereas SVS IV is newly-synthesized and accumulated in the medium of cultured seminal vesicle cells established from androgen primed rats, cultured cells from control rats appear to synthesize and accumulate SVS IV in a precursor form. Results of this work show that seminal vesicle epithelial cells in culture not only retain several structural features representative of the tissue but also serve as a potential system for the study of androgen action.

An Automated Perifusion System for the Study of Rat Spermatogenesis in Vitro

We report details of an automated perifusion system for the study of spermatogenesis in vitro. A potential of this new system is the evaluation of hormone/growth factor effects on the mitotic clonal expansion of spermatogonia and the meiotic and postmeiotic clonal differentiation of spermatogenic cells in coculture with Sertoli cells and in incubated seminiferous tubular segments.

Spermatogenesis In Vitro: Searching for In Vivo Correlates

The development of methodology for the study of spermatogenesis in vitro has been a major task that remained elusive (Wolff and Haffen, 1965; Steinberger, 1975). Significant advances have been reported in the culture of meiotic cells of lilaceous plants (Hotta et al., 1966), and spermatogenic cells of Xenopus (Risley, 1983) and Drosophila (Liebrich, 1981). Attempts to maintain isolated spermatogenic cells viable for long periods of time (more than 48 h) have been relatively unsuccessful. In fact, the seminiferous epithelium contains spermatogenic cells that maintain spatial and functional relationships with Sertoli cells, and this cell-cell interaction is likely to play an important role in spermatogenesis in vivo and in vitro.

Antibodies to rat sperm tail polypeptides recognize Sertoli cell secretory proteins

We have previously reported that (a) polyclonal antisera raised against rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein recognize outer dense fiber polypeptides from rat sperm tail, and (b) protein S70 is antigenically related to polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein. We now report that polyclonal antisera generated against three different outer dense fiber polypeptides recognize (a) the putative antigen of the sperm tail and (b) Sertoli cell secretory protein S70 and its antigenically-related polypeptides. Immunogold electron microscopy shows that outer dense fibers of epididymal sperm crossreact with anti-S70 serum as well as with an antiserum raised against the polypeptide D complex of extracted outer dense fibers. Electron microscopy demonstrates that outer dense fibers consist of filamentous, coil-coiled units aligned side-by-side with each other. Results of this study strengthen the antigenic homology between Sertoli cell secretory proteins and outer dense fiber polypeptides of the sperm tail.