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PubMed1990

How is the flagellar length of mature sperm determined? II. Comparison of tubulin synthesis in spermatids between newt and Xenopus in vitro.

In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster. The average final length of flagella in Xenopus round spermatids was 35 mum, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 mum, almost half that of mature sperm. Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids. The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids. But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species. Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded. Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded. Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids. The period of flagellar elongation almost coincided with the period of tubulin synthesis. The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes. Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.

Acrosome↗

PubMed1998

Microtubule and chromatin organization during the first cell-cycle following intracytoplasmic injection of round spermatid into porcine oocytes.

The objective of this study was to determine microtubule assembly and chromatin configuration in porcine oocytes during the first cell cycle following round spermatid injection into matured porcine oocytes in the presence or absence of electrical stimulation. The oocytes with two large pronuclei and two polar bodies were classified as normal fertilization at 6 to 8 h following injection. The incidence of normal fertilization following round spermatid injection with electrical stimulation was significantly higher (21/45, 47%) than that following injection alone (6/39, 15%). Although a small microtubular aster was organized near the decondensed spermatid chromatin in some oocytes (2/6, 33%, spermatid injection alone; 9/21, 29%, spermatid injection and electrical stimulation), it did not enlarge nor fill the cytoplasm. Instead, a dense network of microtubules in the cytoplasm was organized from cortex. At 12 to 15 h after injection, we classified the oocytes with closely apposed pronuclei as normal fertilization. The electrical stimulation following spermatid injection enhanced (P < 0.05) the incidence of normal fertilization (18/54, 33%) compared with spermatid injection alone (7/52, 13%). During pronuclear movement, the maternally derived microtubules filled the whole cytoplasm, which appeared to move male and female chromatin. Mitosis and two-cell division were observed at 20 to 24 h after spermatid injection with electrical stimulation (12/41, 29%). At mitotic metaphase, the microtubular spindle had focused astral poles, and chromosomes were aligned on the spindle equator. During mitosis, asters were assembled at each spindle pole, and they filled the cytoplasm. These results suggested that round spermatid nuclei of the pig can develop into a morphologically normal pronucleus in matured porcine oocytes and are competent to participate in syngamy with the ootid chromatin. In addition, functional microtubules for complete fertilization with spermatid were not associated with male-derived centrosome but were organized solely from maternal stores.

Animals↗

PubMed1984

Nuclear elongation of dissociated newt spermatids in vitro and their nuclear shortening by antimicrotubule agents.

Dissociated newt spermatids with an initial cell length of 20-35 microns increased in length at an average rate of 35-46 microns during 5 days of culture at 22 degrees C. 10(-5) M vinblastine sulfate shortened the length of nearly all the spermatids of various initial lengths to that of round spermatids within 24 h at 22 degrees C. Application of vinblastine to the spermatids immediately following initiation of nuclear elongation caused the nuclei to become completely round within 1 h. 3 X 10(-6) M colcemid, 10(-4) M colchicine, 2 X 10(-5) M nocodazole and 10(-4) M griseofulvin also shortened the spermatid length. The effects of these five antimicrotubule agents were irreversible. Neither 10(-4) M beta-, gamma-lumicolchicine nor 1.0 micrograms/ml cytochalasin B (CB) had any effect on spermatid elongation. Spermatids incubated at 4 degrees C for 6 days shortened by 20-50%, but after transfer to 22 degrees C they started to elongate. An ultrastructural study showed that during nuclear elongation the number of microtubules increased in proportion to the elongation, and that the microtubules surrounded the whole nucleus from its apical to caudal end. After addition of vinblastine many microtubular crystals appeared in the cytoplasm of the spermatids. It was concluded that microtubules are a prerequisite for nuclear elongation of newt spermatids, and it is speculated that microtubules act directly in the initiation and continuation of the nuclear elongation of newt spermatids.

Animals↗

PubMed1998

Pregnancy and birth after transfer of embryos that developed from single-nucleated zygotes obtained by injection of round spermatids into oocytes.

OBJECTIVE: To use injection of spermatids into oocytes as a mode of infertility treatment in cases in which spermatozoa are not available. DESIGN: Prospective clinical evaluation and case report. SETTING: In Vitro Fertilization Unit, Herzliya Medical Centers, Herzliya-on-Sea, Israel. PATIENT(S): Thirteen couples with male factor infertility in which the male partner lacked spermatozoa in the ejaculate or testicular biopsy samples. INTERVENTION(S): Round spermatid injection and elongated spermatid injection into oocytes. MAIN OUTCOME MEASURE(S): Evaluation of the rate of two-pronucleated and single-nucleated zygote development. RESULT(S): The rate of two-pronucleated zygote development after round spermatid injection and elongated spermatid injection was relatively low (27% and 36%, respectively). Single-nucleated zygotes develop more frequently after round spermatid injection and elongated spermatid injection (35% and 17%, respectively) than after intracytoplasmic sperm injection with mature spermatozoa. A normal pregnancy and childbirth resulted from the transfer of 4 cleaving embryos, each of which developed from a single-nucleated zygote in a round spermatid injection treatment cycle with ejaculated spermatids. CONCLUSION(S): Embryos derived from single-nucleated zygotes after spermatid conception can be viable and give rise to an ongoing clinical pregnancy and childbirth.

Adult↗

PubMed1993

Behaviour of hamster and mouse round spermatid nuclei incorporated into mature oocytes by electrofusion.

Round spermatids of the hamster and mouse were electrofused with homologous mature oocytes to examine the behaviour of their nuclei within the ooplasm. A single spermatid was inserted in the perivitelline space of a mature oocyte and an electric fusion pulse given. In the hamster, the best spermatid-oocyte fusion took place when the oocytes were pretreated with neuraminidase, subjected to 30 s AC (2 MHz, 20 V/cm) followed by a single fusion DC pulse (3000 V-cm, 10 microseconds) and another 30 s AC current. Inclusion of micromolar Ca2+ and Mg2+ in the fusion medium was essential for oocyte activation. Under these conditions all oocytes were activated and 20-40% fused with spermatids. Of these fused oocytes only 5-10% had fully developed spermatid (male) and oocyte (female) pronuclei. In the rest the spermatid-derived pronuclei remained small throughout the pronuclear stage. However, nucleolus-like structures appeared de novo and DNA synthesis occurred in these small pronuclei. Regardless of the size of male pronuclei, chromosomes from the spermatid and oocyte appeared to mingle and participate in the first cleavage. About 70% of the fused oocytes developed into the 2-cell stage. Electrofusion of mouse oocytes with spermatids was less efficient. Even under the best conditions tested, less than 10% of the oocytes fused with spermatids. Here again, most spermatid nuclei remained small throughout the pronuclear stage.

Animals↗

PubMed1984

Generation of flagella by cultured mouse spermatids.

During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained by enzymatic treatments and round spermatids (greater than 90% pure) were purified by unit gravity sedimentation. As determined by Nomarski or phase-contrast microscopy, no cells had flagella immediately after isolation; flagella were first clearly detected after 6 1/2 h of culture in Eagle's minimal essential medium containing 10% fetal bovine serum and 6 mM lactate. After 24 h, approximately 20% of round spermatids had formed flagella. Multinucleated round spermatids often formed multiple flagella, the number never exceeding the number of nuclei per symplast. Round spermatids were the only spermatogenic cells capable of tail formation. Flagella elongation was blocked by 1 microM demecolcine, an inhibitor of tubulin polymerization. Indirect immunofluorescence localized tubulin in the flagella. As seen by scanning electron microscopy, flagella developed as early as 2 h after culture and continued to elongate over the next 20 h, reaching lengths of at least 19 micron. Transmission electron microscopy demonstrated that flagella formed in culture resembled flagella from Golgi-phase round spermatids in situ; the flagella consisted of "9+2" axonemes lacking other accessory structures such as outer dense fibers and the fibrous sheath. As determined by acridine orange staining of the developing acrosomes, all spermatids that formed flagella in culture were Golgi-phase spermatids. By these criteria, the structures are indeed true flagella, corresponding in appearance to what others have described for early mammalian spermatid flagella in situ. We believe this is the first substantiated report of limited in vitro differentiation by isolated mammalian spermatids.

Animals↗

PubMed1998

Is there a future for spermatid injections?

Microinjection of spermatids into oocytes has proven to be a successful assisted reproduction procedure in the animal model. In the human, low fertilization and cleavage to the 4-cell stage were reported after intracytoplasmic sperm injection (ICSI) with round spermatids. In comparison with a conventional ICSI-testicular sperm extraction (TESE) programme, the implantation rate after round spermatid injection is dramatically low. Different problems have been encountered during the development of the spermatid injection technique and they could be partially responsible for the lower outcome when using round spermatids. Compared with the round spermatid cells, spermatids in the elongation phase are easy to isolate and identify from other round cells present in a wet preparation. The morphological identification does not reveal anything about the viability or the genetic normality of the round spermatids. Severe testicular dysfunction may have consequences on the quality of the few spermatogenic cells present. Others factors, such as the pathology of the patient, play an important role in the successful treatment. Even if the results are extremely low, spermatid injection seems more favourable for men who have already proven their capacity to produce some spermatozoa. A spermatogenic block at the round spermatid level has led to early abortions, increasing the suspicion of the role of a genetic factor. In order for this technique to be safe for use in clinics, more intensive work is needed to improve the selection and handling of cells and to ascertain the genomic imprinting and gene expression necessary for embryonic development. Hence, when using immature cells for conception, the screening of the patient and the follow-up of the pregnancies and babies should be mandatory.

Animals↗

PubMed1999

Influence of the male reproductive tract on the reproductive potential of round spermatids abnormally released from the seminiferous epithelium.

Round spermatids can be collected from testicular biopsy material or occasionally from semen samples. We evaluated the influence of the passage of round spermatids through the male reproductive tract on their reproductive potential. A model of abnormal release of round spermatids from the seminiferous epithelium was created in mature male rats (group A). Additional sham-treated rats of the same age served as a control group (group B). Round spermatids were collected from the testicles of rats of both groups, the epididymides of rats of group A, and the vaginae of mature female rats mated with rats of group A. Isolated round spermatids were processed for ooplasmic injections. Injected oocytes were cultured. At 96 h post-injection, the blastocyst development rate was significantly higher in the groups of oocytes injected with testicular spermatids than the groups of oocytes injected with spermatids recovered from the vaginae, or the head, body, or tail of the epididymides. It appears that round spermatids recovered from testicular biopsy material have larger reproductive capacity than ejaculated round spermatids, due to mechanical or chemical detrimental influences of storage/passage through the male reproductive tract (outside the testicle) on the capacity of round spermatids to induce optimal early embryonic development.

Animals↗

PubMed1996

Chromatin reorganization in rat spermatids during the disappearance of testis-specific histone, H1t, and the appearance of transition proteins TP1 and TP2.

Transition proteins replace testis-specific histones and are finally replaced by protamines in the nucleus of germ cells during spermiogenesis. In this study, immunoperoxidase and immunogold localization were used to determine both qualitatively and quantitatively the intracellular distribution of testis-specific histone (H1t), transition protein 1(TP1), and transition protein 2 (TP2) during rat spermatogenesis. H1t labeling was concentrated over heterochromatin in the nucleus of late-pachytene spermatocytes and spermatids up to mid-steps 10. In step 9 spermatids, H1t was confined to the caudal end of the nucleus where heterochromatin was still present, while in early step 10 spermatids, only a few of the nuclei remained caudally labeled. In late step 10 spermatids, a fibrillar chromatin network was distributed throughout the nucleus coincident with the loss of H1t. A statistically significant rise in TP1 and TP2 labeling density over control values was first encountered in the nucleus of step 11 spermatids coincident with the initiation of condensation of the fibrillar chromatin. The TP1 and TP2 labeling density progressively increased in nucleus of step 11-13 spermatids with the apical to caudal condensation of the fibrillar chromatin, In step 13 spermatids, the chromatin was homogeneously condensed throughout the nucleus. In the case of TP1, the nuclear labeling density gradually declined after step 13 and disappeared by step 17. In the case of TP2, the nuclear labeling density disappeared by step 16. This study shows that, coincident with the loss of H1t, the chromatin of the spermatid is reorganized into a fibrillar network, whereas, coincident with the appearance and progressive increase of TP1 and TP2, the fibrillar chromatin condenses in an apical to caudal direction in the nucleus of the spermatid. Thus the remodeling of chromatin structure during spermiogenesis appears to be a two-step process that is sequentially influenced by the loss of spermatid-specific histones and the appearance of transition proteins.

Animals↗

PubMed2006

The Sertoli-spermatid junctional complex adhesion strength is affected in vitro by adjudin.

The actin-based cell-cell adherens junction (AJ) between the Sertoli cell and the germ cell in the mammalian testis is important not only in mechanical adhesion of the cells, but in the morphogenesis and differentiation of the germ cells. The Sertoli ectoplasmic specialization (ES), a specialized type of AJ, is associated with Sertoli-spermatid binding and is important in cell-cell adhesion in the seminiferous epithelium. Abnormal or absent Sertoli ESs have been associated with step-8 spermatid sloughing and oligospermia in conditions associated with reduced fertility potential. The reproductive hormones, follicle stimulating hormone (FSH), and testosterone (T) have also been shown to play a role in the regulation of binding of spermatids at the Sertoli-spermatid junctional complex (STJC). Adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide] is a potential male contraceptive and is thought to exhibit its contraceptive effects by interrupting the STJC. It has been shown that this compound induces reversible germ cell loss from the seminiferous epithelium, particularly elongating/elongate/round spermatids and spermatocytes. Using a micropipette pressure transducing system (MPTS) to measure the force needed to detach step-8 spermatids from Sertoli cells, this study examined the strength of the STJC in Sertoli-spermatid cocultures in the presence of Adjudin (1 ng/mL, 50 ng/mL, 125 ng/mL, or 500 ng/mL in EtOH) and hormones [FSH (0.1 micro g/mL, NIDDK-oFSH-20, AFP7028D, 175 x NIH-FSH-S1), T (100 nM)] to optimize in vitro binding. The average forces required to detach the spermatids from the underlying Sertoli cells in the presence of 1 ng/mL, 50 ng/mL, 125 ng/mL, and 500 ng/mL Adjudin were 18.2 x 10(-10) pN, 14.3 x 10(-10) pN, 7.74 x 10(-10) pN, and 6.51 x 10(-10) pN, respectively. The average force required to detach step-8 spermatids in the presence of vehicle only (control) was 19.0 x 10(-10) pN. A significant difference for Adjudin concentrations at or above 125 ng/mL was determined by one-way ANOVA (P < .05). These data confirm that Adjudin is effective in reducing the strength of the STJC, identifying Adjudin as a potential contraceptive agent in the male by inducing spermatid sloughing and therefore oligospermia.

Adherens Junctions↗

PubMed1998

Spermatids as gametes: indications and limitations.

The feasibility of achieving viable embryos, developing to term after transfer into the uterus, by fertilizing oocytes with spermatids has been demonstrated both in animal studies and in preliminary human clinical trials. Here we review the current clinical indications of spermatid conception and discuss the predictable success rates associated with each of these indications. Potential health hazards relating to the use of spermatids for conception are updated taking into account the risk of abnormal or incomplete epigenetic modifications of newly discovered human imprinted genes. We also add new experimental data showing the occurrence of spermatids in patients lacking spermatozoa and demonstrating that round spermatids recovered from patients with complete spermiogenesis failure (no elongated spermatids or spermatozoa ever detected in the patient's history) are often deficient in the factor(s) responsible for oocyte activation. The possible consequences of this deficiency for the occurrence of abnormal fertilization patterns and for the impairment of further preimplantation and post-implantation development are discussed. It is concluded that the development of diagnostic tests to assess the intrinsic quality of spermatids, with regard to their ability to act as gametes, is urgently needed as part of pre-treatment diagnosis before infertile couples are included in a spermatid conception programme. Centres wishing to use spermatids in human assisted reproduction should also be prepared to offer adequate diagnostic methods to control genomic imprinting abnormalities in the progeny.

Female↗

PubMed2004

Appearance of the oocyte activation of mouse round spermatids cultured in vitro.

This study was undertaken to determine the expression time of fertilization and oocytes activation abilities of spermatids in the mouse. When elongating or elongated spermatids isolated from fresh testes of adult males (B6D2F1) were injected into mature mouse oocytes, both spermatids could activate the mature oocytes and occur fertilization. On the one hand, the round spermatids could not activate mature oocytes, when microinjected into oocytes. In some experiments, recovered round spermatids were cultured under co-culture systems using Sertoli cells as a feeder cell. Under the co-culture system, developed elongating spermatids could stimulate and fertilized mature oocytes. These results indicate that the start of oocyte activation appearance is between the stage of round spermatid and elongating spermatids and the activation ability increases with the advance of spermiogensis. On the other hand, round spermatids isolated from males of ICR strain mouse already have the oocyte activation ability and the fertilizing ability. The result obtained suggests that the expression time of the oocyte activating ability is difficult between the mouse strain.

Animals↗

PubMedDate not supplied

Strength measurement of the Sertoli-spermatid junctional complex.

The Sertoli cell ectoplasmic specialization (ES) is a specialized domain of the calcium-dependent Sertoli cell-spermatid junctional complex. Not only is it associated with the mechanical adhesion of the cells, but it also plays a role in the morphogenesis and differentiation of the developing germ cells. Abnormal or absent Sertoli ESs have been associated with step-8 spermatid sloughing and subsequent oligospermia. With a micropipette pressure transducing system (MPTS) to measure the force needed to detach germ cells from Sertoli cells, this study examined, for the first time, the strength of the junction between Sertoli cells and spermatids and between Sertoli cells and spermatocytes. The mean force needed to detach spermatocytes from Sertoli cells was 5.25 x 10(-7) pN, prestep-8 spermatids from Sertoli cells was 4.73 x 10(-7) pN, step-8 spermatids from Sertoli cells was 8.82 x 10(-7) pN, and spermatids plus EDTA was 2.16 x 10(-7) pN. These data confirm the hypothesis that step-8 spermatids are more firmly attached to Sertoli cells than are spermatocytes and pre-step-8 spermatids and that calcium chelation reduces binding strength between Sertoli cells and spermatids. The MPTS is a useful tool in studying the various molecular models of the Sertoli-germ cell junctional strength and the role of reproductive hormones and enzymes in coupling and uncoupling of germ cells from Sertoli cells.

Animals↗

PubMed2000

Round spermatid separation and in-vitro maturation.

OBJECTIVE: To combine 2 methods; spermatid isolation and in-vitro maturation to improve their quality and enhance their ability of fertilization. METHODS: A discontinuous Percoll gradient was used to separate immature germ cells. Co-culture with Vero cells was attempted to convert round spermatids into more mature forms. In total 87 spermatids were studied. RESULTS: Of a final number of 77 round spermatids only 12 (15.5%) showed a certain degree of maturation in 3 out of 7 patients (42%). Of those 12 maturing spermatids, only 4 developed to an early elongated spermatid stage Sc1, but without flagella. CONCLUSION: Considering the limited in-vitro round spermatid maturation achieved in this study, the low fertilization and pregnancy rate with spermatids in general and round spermatids in particular, further refinements of this technology have to be achieved before its regular implementation in routine clinical practice is justifiable.

Animals↗

PubMed1976

Anchoring device between Sertoli cells and late spermatids in rat seminiferous tubules.

Near the end of spermiogensis, the late spermatids remain attached to the superficial layer of the seminiferous epithelium for an appreciable period of time (i.e., 3 to 4 days). Ths sickle-shaped heads of the spermatids are embedded in an apical process of Sertoli cell cytoplasm which is connected to the rest of the cell by a narrow stalk. In the concavity of the head several long (2-3 mum) and very narrow (50 nm) tubular projections of the spermatid's plasma membrane invaginate the Sertoli cell cytoplasm. These tubular processes terminate by a bulbous swelling which may measure up to 1 mum in diameter. Along the process the plasma membrane of the Sertoli cell is closely apposed to the spermatid's membrane, the intracellular space being only 6-8 nm wide. In the Sertoli cytoplasm immediately surrounding the tubular portion of the structure there is an accumulation of filamentous material, while next to the bulbous extremity there are, at a shrot distance, smooth surfaced cisternae of endoplasmic reticulum. The whole structure was referred to as a tubulobulbar complex. These complexes, of which there are up to 24 per spermatid, appear as these cells complete their migration toward the apex of the Sertoli cells. They disappear just before the release of the spermatids in the lumen of the seminiferous tubule as a result of the fragmentation of the spermatid's plasma membrane followed by a resorption of the Sertoli plasma membrane. Morphological evidence suggests that the Tubulobulbar complexes serve as anchoring devices that retain the spermatids at the surface of the seminiferous epithelium while their dissolution contributes in part to the process of spermiation. Similar tubulobulbar complexes were also formed by the plasma membranes of two adjacent Sertoli cells close to the Sertoli-Sertoli tight junctions near the tubular limiting membrane.

Animals↗

PubMed1986

Distribution of actin in Sertoli cell ectoplasmic specializations and associated spermatids in the ground squirrel testis.

We have investigated the possibility that the complex patterns of fluorescence associated with spermatids of the ground squirrel labeled with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) are due to the presence of filamentous actin within the spermatids themselves rather than to actin in attached Sertoli cell ectoplasmic specializations, as previously reported (J. Cell Biol., 100:814-825). Enzymatic treatments (trypsin, DNAase 1) freed Sertoli cell ectoplasmic specializations from spermatids and resulted in a loss, from the spermatids, of the complex fluorescence patterns, suggesting that the latter were generated by labeled actin in ectoplasmic specializations. Moreover, ectoplasmic specializations that were detached enzymatically from spermatids demonstrated the same fluorescence patterns as those emitted from spermatids in the intact or mechanically fragmented seminiferous epithelium. Most spermatids, however, do display a weak and diffuse pattern of fluorescence that changes during spermatogenesis and that is localized between the acrosomal cap and nucleus. S-1 decoration confirmed this subacrosomal localization and further demonstrated that the actin in adjacent Sertoli cell ectoplasmic specializations is arranged in a unipolar fashion. We conclude that the complex patterns of actin fluorescence associated with mechanically isolated spermatids are a superimposition of both Sertoli cell and germ cell actin; however, the latter is either poorly detected or not detected at all when Sertoli cell ectoplasmic specializations overlie the germ cells.

Actins↗

PubMed1991

Distribution of actin isoforms within cells of the seminiferous epithelium of the rat testis: evidence for a muscle form of actin in spermatids.

Recently, a cDNA that coded for an enteric smooth muscle gamma-actin (SMGA) that was expressed in post-meiotic mouse testicular cells was identified. To determine the cellular location(s) of the protein encoded by this cDNA, this SMGA was probed for by immunocytochemistry in the cells of the seminiferous epithelium with two different monoclonal antibodies (Mabs), B4 and HUC 1-1, known to be muscle actin selective. As a control, we also examined the immunoreactivity of a third Mab, C4, that reacts with all non-muscle and muscle vertebrate isoactins. Using light and electron microscopy, a progressive increase in immunolabeling was observed with the muscle selective HUC 1-1 Mab over a loose actin filamentous network distributed throughout the cytoplasm of steps 4-16 spermatids. Thereafter, the labeling decreased such that at step 17 spermatids, only cytoplasmic labeling in the tail of the spermatids was observed. No labeling of this network was noted with the C4 or B4 Mabs. However, myoid cells enveloping seminiferous tubules and smooth muscle cells of interstitial blood vessels demonstrated comparable intense labeling with each of the three Mabs. The C4 Mab intensely labeled actin filaments of the Sertoli-Sertoli and Sertoli-spermatid ectoplasmic specializations. Also well labeled were numerous actin filaments found in the apical Sertoli cell processes encapsulating the heads of late step 19 spermatids at stage VII of the cycle of the seminiferous epithelium. In addition, actin filamentous bundles enveloping tubulobulbar complexes of the late spermatids within the Sertoli cell apical processes were intensely labeled. The actin filaments in the Sertoli apical processes and surrounding the tubulobulbar complexes were also strongly immunolabeled with the HUC 1-1 Mab. The C4 Mab but not the B4 or HUC 1-1 Mabs, recognized actin in the subacrosomal space of steps 4-18 spermatids. This study suggests that there are muscle isoforms of actin within the cytoplasm of developing spermatids and within apical processes of Sertoli cells.

Actins↗

PubMed1976

Haploid genome reactivation and recovery by cell hybridization. Induction of DNA synthesis in spermatid nuclei.

DNA replication in haploid spermatid nuclei has been induced by hybridization of mouse early spermatids to proliferating HeLa cells. Use of polyethylene glycol rather than inactivated Sendai virus as the cell fusion agent was found to be essential to the production of large numbers of heterokaryons containing spermatid nuclei. DNA replication was detected in the heterokaryons by autoradiography. Density of silver grains over spermatid nucleic closely approximated the grain density over labelled HeLa nuclei in the same heterokaryons. Mouse centromeric heterochromatin appeared to be labelled last during the spermatid DNA synthetic period. On the average, HeLa nuclei in heterokaryons began DNA synthesis before spermatid nuclei. Results indicated, however, that DNA synthesis by HeLa nuclei might not be a prerequisite for spermatid DNA synthesis. These experiments demonstrate induction of DNA synthesis in spermatid nuclei, the first major step toward reactivation and recovery of their haploid genome by cell hybridization.

Animals↗