Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Spermatids”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources
PubMed1990

Ethanol-phosphotungstic acid and bismuth staining of spermatid nucleoli in mouse spermiogenesis.

The nucleoli of developing mouse spermatids were examined with ethanol-phosphotungstic acid (E-PTA) staining, and also with bismuth staining following formaldehyde fixation (FA-Bi staining) and glutaraldehyde fixation (GA-Bi staining). Only the cortical zone of the nucleolar dense fibrillar component (DFC) in the round spermatids was stained with E-PTA, while the inner area remained either faintly (early Golgi-phase spermatids) or completely unstained (cap-phase spermatids). Incubation of the fixed testis with dithiothreitol before E-PTA staining resulted in homogeneously intense staining of the DFC. The facts suggest that numerous E-PTA-positive basic proteins were present in the DFC, but disulfide crosslinks formed in the DFC proteins prevent penetration of PTA into the DFC interior. The DFC was stained with bismuth after FA-Bi and GA-Bi staining until the disappearance of the nucleoli occurring in acrosome-phase spermatids. The fibrillar center, homogeneously stained using E-PTA, FA-Bi, and GA-Bi methods was present in the nucleoli of Golgi-phase and early cap-phase spermatids, but disappeared in the nucleoli of late cap-phase spermatids. These results are discussed based on the previous studies dealing with the ribosomal RNA synthesis in mouse spermiogenesis.

Animals↗
PubMed2006

The 5' UTR and 3' UTR of the sperm mitochondria-associated cysteine-rich protein mRNA regulate translation in spermatids by multiple mechanisms in transgenic mice.

The Smcp mRNA encoding the sperm mitochondria-associated cysteine-rich protein is translationally repressed in round spermatids and translationally active in elongated spermatids. The patterns of transcription and translation of fusions of the Smcp promoter, the green fluorescent protein coding region (Gfp) and various combination of the Smcp and Gfp 5' UTR and 3' UTR have been studied in transgenic mice. 518 nt of Smcp 5' flanking region and 8 nt of 5' UTR drive transcription of mRNAs containing the Gfp coding region in early round spermatids at the same transcription start site as the natural Smcp gene. Transcripts containing both the Gfp 5' and 3' UTRs are translationally active in step 2 spermatids as detected by GFP fluorescence in squashes of living seminiferous tubules from adult testes, and the presence of polysomal mRNAs in sucrose gradient analyses of testes from 21-day-old prepubertal mice, which contain early round spermatids and lack elongated spermatids. By comparison, expression of GFP is delayed until steps 5 and 10, respectively, in transcripts containing the Smcp 5' UTR and Gfp 3' UTR and the Gfp 5' UTR and the Smcp 3' UTR. Sucrose gradient analysis of 21-day-old testes demonstrates that transcripts containing the Smcp 3' UTR exhibit a bimodal distribution in free-mRNPs and polysomes, indicating that the 3' UTR blocks the expression of GFP after the transcripts have entered the elongation phase, a mechanism that may involve microRNAs. The Smcp 5' UTR reduces the levels and size of polysomes in adult testis. In addition, the natural Smcp mRNA contains a positive control element that counteracts the inhibition of translation by the Smcp 5' UTR in adult testis, and the Smcp 3' UTR strongly localizes GFP fluorescence in step 10 spermatids.

3' Untranslated Regions↗
PubMed2006

FNDC3A is required for adhesion between spermatids and Sertoli cells.

Symplastic spermatids (sys) male mice are sterile due to a recessive mutation that causes defective adhesion between spermatids and Sertoli cells within the seminiferous epithelium. We show that the mutation in sys mice involves a deletion of 1.24 Mb of chromosome 14. Comparative genomic analysis suggests that this region contains only one gene, Fndc3a. A genetic complementation analysis using mice with a specific mutation within Fndc3a verifies that mutation of Fndc3a is the cause of male sterility in sys mice. Fndc3a is a member of a three-gene family in mice. Fndc3a, which is expressed in several tissues including testis, encodes a novel protein composed of a proline-rich amino-terminus, nine fibronectin type-III domains, and a hydrophobic carboxy-terminus. The proline-rich region of each family member contains conserved amino acids that include a PPGY consensus binding site for type I WW domain containing proteins. The hydrophobic carboxy-terminus is similar to that found in 'tail-anchored' proteins, integral membrane proteins that are localized to the cytosolic face of the endoplasmic reticulum. Immunohistochemical staining indicated that FNDC3A localizes to the acrosome of spermatids, as well as to Leydig cells in the mouse testis. Acrosomal localization of FNDC3A is observed in spermatids between step 2 and step 10 inclusive. In step 12 spermatids, FNDC3A is largely absent from the acrosomal region with immunostaining being localized to vesicular structures located within the cytoplasm of elongate spermatids. Models are presented for the function of FNDC3A in mediating spermatid-Sertoli adhesion during mouse spermatogenesis.

Alleles↗
PubMed2000

Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells.

OBJECTIVE: To examine whether the results of assisted reproduction with the use of elongated spermatids from a man with incomplete arrest of spermiogenesis and a high frequency of apoptosis among postmeiotic germ cells can be improved by germ cell in vitro culture. DESIGN: Case report. SETTING: Private assisted reproduction centers and a university department. PATIENT(S): Man with incomplete spermiogenesis failure. INTERVENTION(S): Testicular spermatid extraction, in vitro culture of testicular biopsy samples, intraoocyte injection of elongated spermatids, embryo culture, and transfer. MAIN OUTCOME MEASURE(S): Fertilization rate, embryo morphology, and pregnancy. RESULT(S): An assisted reproduction attempt with viability-selected elongated spermatids from a fresh testicular biopsy sample resulted in a poor fertilization result and embryo quality. Further analysis of the sample used in this attempt showed a high incidence of apoptosis among postmeiotic germ cells. A second attempt was then performed with in vitro culture of testicular cells for 24 hours before spermatid injection. This procedure led to a significant decrease in the frequency of apoptosis among viability-selected spermatids, to improvement of the fertilization rate and embryo quality, and to the birth of healthy twins. CONCLUSION(S): In vitro culture of testicular biopsy samples before assisted reproduction with elongated spermatids may improve the efficacy of treatment in cases of massive in vivo apoptosis of postmeiotic germ cells.

Adult↗
PubMed1998

Immature spermatids are not prevalent in semen from men who are receiving androgen-based contraceptive regimens.

OBJECTIVE: To determine whether immature spermatids increase in semen in response to hormonal contraceptive treatments. Such a finding would support the existence of a defect in spermiogenesis, which in turn may explain the reported variability in sperm output. DESIGN: Semen smears were obtained from healthy men undergoing randomized control trials of T plus progestin contraceptive treatments. PATIENT(S): Healthy men (21-49 years) with normal semen analyses received T (50-100 mg IM weekly) in combination with either desogestrel (150-300 microg daily, n = 5) or levonorgestrel (125-250 microg daily, n = 10) for 24 weeks. Semen smears were made during spermatogenic suppression and recovery. Nine control subjects were also assessed. MAIN OUTCOME MEASURE(S): Semen analyses were performed using World Health Organization criteria. Immature spermatids and white blood cells in semen were identified by immunostaining with monoclonal antibodies to the human intra-acrosomal antigen SP-10 and the ubiquitous white cell CD-45 antigen, respectively. RESULT(S): In a total of 14 normal ejaculates (9 control and 5 pretreatment) 74+/-14 million/mL sperm (mean+/-SEM) were seen together with a few immature spermatids (0.69+/-0.20 million/mL). During contraceptive treatments, spermatid number decreased in parallel with the sperm concentration and spermatids disappeared in most subjects. No significant changes were seen in either leukocyte or immunonegative round cell concentration (0.41+/-0.25 and 0.25+/-0.09 million/mL in controls, respectively) in response to treatments. CONCLUSION(S): Spermatid sloughing, as assessed by the ejaculation of immature spermatids, is not a feature of T-induced spermatogenic regression in men; rather, the decline in both mature and immature germ cells in the ejaculate probably results from a decline in the number of precursor cells, ultimately resulting in severe oligo- or azoospermia. Detailed studies on the sites of spermatogenic interruption are required to understand the variability in responses seen after contraceptive therapies in men.

Adult↗
PubMed1997

Further observations on carbendazim-induced abnormalities of spermatid morphology in rats.

The effects of a microtubule poison, carbendazim, on rat spermiogenesis were examined for abnormalities of nuclei, acrosome and manchette in round and elongating spermatids in Stages VII-XII on days 7.5, 9.5, 10.0 and 10.5 post-treatment using routine electron microscopy. Spermatid nuclear abnormalities were observed in Stages IX-XI on day 9.5 and at greater post-treatment intervals. Nuclear abnormalities included nuclear distortions, various types of nuclear invaginations and abnormal positioning of the modified nuclear envelope. Acrosomal abnormalities were noted on day 7.5 and at greater intervals. Discontinuous, multiple granular and fragmentary acrosomes were observed in Stages VII-XI. In addition, spermatids with complete absence of acrosome (acrosome-deficient spermatids) were observed in Stages VII-X. Poorly-formed and absent ectoplasmic specializations were seen in the cytoplasm of Sertoli cells next to the acrosome-deficient spermatids. A major abnormality of the manchette was irregular positioning of the manchette microtubules in steps 9-11 spermatids on day 9.5 and at greater intervals, which resulted in nuclear invagination. The results indicate that carbendazim induces abnormalities in spermatid morphology that are common to those reported in testes treated with several chemical compounds and in testes of mutant animals.

Acrosome↗
PubMed1997

Characterization of nerve growth factor precursor protein expression in rat round spermatids and the trophic effects of nerve growth factor in the maintenance of Sertoli cell viability.

Nerve growth factor (NGF) is expressed by rat round spermatids and is thought to participate in the paracrine regulation of spermatogenesis. In order to elucidate the role of NGF in the rat testis, we further characterized the NGF immunoreactive protein secreted by round spermatids and examined the effect of NGF beta and related neurotrophin family members on the maintenance of Sertoli cell viability. Round spermatids were isolated from rat testes by centrifugal elutriation and the conditioned medium dialyzed/concentrated for the preparation of round spermatid protein (RSP). Immunoblot analysis of RSP with anti-NGF beta antibody identified two immunoreactive bands of 31 and 22 kD, whereas the 13 kD mature form of NGF beta was not observed. Similarly, immunoblot analysis of RSP with an antibody raised against a synthetic peptide (L38) corresponding to the -3 to -40 sequence of proNGF also recognized two immunoreactive bands of 31 and 22 kD. These results are consistent with the identification of two NGF precursors. Interestingly, immunoblot analysis of RSP with an antibody raised against a synthetic peptide (N4) corresponding to the -71 to -46 sequence of proNGF only recognized one immunoreactive band of 31 kD, consistent with the larger NGF precursor observed with the L38 antibody. In a bioactivity test of PC-12 neurite outgrowth, the 31 kD NGF precursor induced flattening and neurite outgrowth of PC-12 cells, consistent with NGF bioactivity. The 22 kD NGF precursor induced modest and inconsistent neurite outgrowth. These results suggest that round spermatids express the 31 and 22 kD precursor forms of the NGF gene product, and that processing of this gene product is incomplete such that the 13 kD mature form of NGF beta is not observed. In view of the role of round spermatids in the paracrine regulation of spermatogenesis, we examined the effect of RSP on the rescue of the viability of Sertoli cells cultured under serum deprived conditions. In the absence of serum, RSP was able to extend the viability of Sertoli cells, and the elimination of this activity by anti-NGF antibody immunoprecipitation of RSP suggests that the NGF precursors in RSP support the maintenance of Sertoli cell viability. In addition, treatment with exogenous NGF beta was able to rescue Sertoli cell viability, whereas L38 peptide, N4 peptide, or the neurotrophins, brain derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 were unable to rescue Sertoli cell viability above control levels. Hence, it appears that round spermatids express the precursor forms of the NGF gene product, but not the mature form of NGF beta, and that the NGF beta moiety of the NGF precursor proteins exhibits trophic activity in the rescue of Sertoli cell viability, consistent with the paracrine regulation of spermatogenesis.

Animals↗
PubMed1988

A cytochemical study of the transcriptional and translational regulation of nuclear transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids.

Immunocytochemical localization and in situ hybridization techniques were used to investigate the presence of spermatid nuclear transition protein 1 (TP1) and its mRNA during the various stages of spermatogenesis in the rat. A specific antiserum to TP1 was raised in a rabbit and used to show that TP1 is immunologically crossreactive among many mammals including humans. During spermatogenesis the protein appears in spermatids as they progress from step 12 to step 13, a period in which nuclear condensation is underway. The protein is lost during step 15. An asymmetric RNA probe generated from a TP1 cDNA clone identified TP1 mRNA in late round spermatids beginning in step 7. The message could no longer be detected in spermatids of step 15 or beyond. Thus, TP1 mRNA first appears well after meiosis in haploid cells but is not translated effectively for the several days required for these cells to progress to the stage of chromatin condensation. Message and then protein disappear as the spermatids enter step 15. In agreement with a companion biochemical study (Heidaran, M.A., and W.S. Kistler. J. Biol. Chem. 1987. 262:13309-13315), these results establish that translational control is involved in synthesis of this major spermatid nuclear protein. In addition, they suggest that TP1 plays a role in the completion but not the initiation of chromatin condensation in elongated spermatids.

Animals↗
PubMed1998

Successful pregnancy after spermatid injection.

We present nine cases of spermatid intracytoplasmic injection for the treatment of non-obstructive azoospermia. In eight cases, no elongated spermatids or spermatozoa were found in previous spermiograms or testicular biopsies. In these patients, treatment was performed using ejaculated (n = 6) and testicular (n = 2) retrieved round spermatids (Sa type). In cases where ejaculated round spermatids were used, they were isolated on the day before oocyte retrieval and left in culture for 24 h before intracytoplasmic sperm injection (ICSI). No pregnancy was obtained in either group, although culturing seemed to increase the fertilization rate. In one other case, elongated spermatids were observed in the previous spermiogram and thus a normal ICSI procedure was scheduled. However, on the day of oocyte retrieval, no spermatids could be recovered from fresh sequential ejaculates, and a testicular open biopsy was then performed. Both round and elongated spermatids were found in the testicular tissue, but only the more mature germinal cells (Sb2) were injected. From this case, a normal pregnancy was obtained which resulted in the birth by Caesarean section at 37 weeks of gestation of a normal healthy baby girl, weighing 2700 g.

Adult↗
PubMed1999

Flow cytometry isolation and reverse transcriptase-polymerase chain reaction characterization of human round spermatids in infertile patients.

Flow cytometry coupled to cell sorting is proposed as a method to isolate round spermatids from testicular biopsies in obstructive azoospermic patients. The cells were separated on the basis of their size and density only. We obtained homogenous populations of alive round spermatids free of lymphocytes and diploid germ cells. The detection of protamine 1 gene (PRM1) and PRM2 expression in the sorted cells proves that these cells are round spermatids. On the contrary, neither the expression of CD3-delta, which is specific to lymphoid cells, nor that of MAGE1, which has been demonstrated in diploid germ cells, could be observed in the round spermatid population even after using a nested polymerase chain reaction (PCR) assay. The flow cytometry procedure failed to isolate round spermatids from ejaculates in non-obstructive azoospermic patients. In > 39 ejaculates tested by reverse transcriptase-PCR, only nine revealed the presence of some round spermatids, as demonstrated by the expression of PRM1. However, these round spermatids did not express PRM2.

Adult↗
PubMed1999

Mouse spermatid nuclei can support full term development after premature chromosome condensation within mature oocytes.

The nucleus of round spermatids, the earliest haploid male germ cells, can participate in the formation of normal zygotes when incorporated into activated oocytes. In this study, we injected mouse round spermatids into homologous mature oocytes that were kept arrested at metaphase II to induce premature chromosome condensation (PCC) of the spermatid nuclei. After full condensation of the spermatid chromosomes, the oocytes were activated by Sr2+-containing medium, into which cytochalasin B was added to prevent extrusion of the segregated female and male chromosomes as polar bodies. Out of 142 oocytes examined, 104 (73%) formed two male (pseudo)pronuclei and two female pronuclei. To restore the diploid state of these zygotes, one of the female pronuclei was removed. When cultured in vitro for 72 hours, all (n = 37) of the constructed embryos developed to the morula/blastocyst stage. When 2-cell embryos and morulae/blastocysts were transferred into pseudopregnant females, 14 (13/96) and 24% (9/37), respectively, developed into term offspring. This study indicates that the spermatid chromosomes, which had undergone PCC, moved safely to opposite poles after oocyte activation. Since round spermatids contain no (in the mouse) or little (in patients with spermatogenic failure) oocyte-activating factor, this method may be used to rescue oocytes that fail to be activated at the time of spermatid injection.

Animals↗
PubMed1999

Evaluation of the fertilization potential of freshly isolated, in-vitro cultured and cryopreserved human spermatids by injection into hamster oocytes.

The clinical potential for fertilization was examined by using the human sperm-hamster oocyte assay system after microinjection of round (RS), elongating (ES) or elongated (EtedS) spermatids retrieved from obstructive and non-obstructive azoospermic patients. Freshly isolated, in-vitro cultured and cryopreserved spermatids were utilized. For each category of microinjected spermatids, we demonstrated that the more mature the injected spermatid, the higher the incidence of fertilization (for freshly isolated spermatids, P < 0.006 and P < 0.008, for in-vitro cultured spermatids, P < 0.007 and P < 0.007 and for cryopreserved spermatids, P < 0.006 and P < 0.007 for obstructive and non-obstructive azoospermic patients respectively). Short term in-vitro culture of the spermatogenic cells did not improve the incidence of fertilization. However, cryopreservation significantly decreased (P < 0.001) the incidence of fertilization when each corresponding spermatogenic cell stage was compared. The incidence of fertilization was not statistically different when corresponding stages of spermatogenic cells were compared from obstructive and non-obstructive patients.

Animals↗
PubMed1999

Fertilization, embryonic development, and offspring from mouse eggs injected with round spermatids combined with Ca2+ oscillation-inducing sperm factor.

Round spermatids, precursor male gametes, are known to possess the potential to achieve fertilization and embryonic development when injected into eggs. However, injection of spermatids alone seldom activates eggs in the mouse, as spermatids by themselves cannot induce an increase in intracellular Ca2+, a prerequisite for egg activation. We injected a mouse round spermatid into an egg simultaneously with partially purified sperm factor from differentiated hamster spermatozoa. The combined injection produced repetitive Ca2+ increases (Ca2+ oscillations) lasting for at least 4 h as observed at fertilization, and induced activation in 92% of eggs. This method provided 75% fertilization success associated with male and female pronucleus formation and development to 2-cell embryos, while only 7% of eggs were fertilized by injection of a spermatid alone. Of the 2-cell embryos, approximately 50% developed to blastocysts during 5 days of culture in vitro, while no blastocysts were obtained following injection of sperm factor alone. Furthermore, the 2-cell embryos, that were created by spermatids and sperm factor and transplanted into foster mothers, developed into normal offspring, although the percentage was only 22%. All infants grew into healthy adults carrying normal chromosomes. The sperm factor served as a complementary factor for successful fertilization by round spermatid injection.

Animals↗
PubMed1996

Spermatid injection into human oocytes. II. Clinical application in the treatment of infertility due to non-obstructive azoospermia.

We have reported recently the first birth after intrauterine transfer of embryos obtained by injection of round spermatids into oocytes in cases of unexpected azoospermia. Here we provide a complete documentation of the series of 11 cases in which this novel method of infertility treatment was employed. In four of these cases, elongated spermatids were identified in the ejaculate, and it was decided to perform elongated spermatid injection (ELSI). In the other six cases, only round spermatids were present, and round spermatid injection (ROSI) was done. In one case, ROSI was given preference to ELSI because of a very poor viability status of elongated spermatids present in the ejaculate. Fertilization of at least one oocyte was achieved in 10 of the 11 treatment cycles; the fertilization rate in these 10 cycles ranged between 7 and 100% with a mean value of 45%. All of the two-pronucleated zygotes cleaved and were transferred to the patient's uterus. A singleton pregnancy was achieved in two ROSI cycles. Both pregnancies developed uneventfully and resulted in the birth of normal infants. These data show the intra-ooplasmic injection of spermatids obtained from the ejaculate may become the treatment of first choice in patients with non-obstructive azoospermia.

Adult↗
PubMed1995

Stage-specific distribution of the spermatid-specific histone 2B in the rat testis.

Male germ cells contain a number of histone variants, most of which are synthesized during either the mitotic or the meiotic stages of spermatogenesis. A spermatid-specific H2B (ssH2B) variant has been identified in mouse round spermatids that has an additional 12 amino acids at its carboxyl terminus as compared to somatic H2Bs. Until now, the presence of this protein in other mammals has not been known. Northern blot analysis using an oligonucleotide probe complementary to a unique coding region of the C-terminus of mouse ssH2B showed that mRNA encoding this protein was present in isolated rat round spermatids. Furthermore, immunoblot analysis of basic nuclear proteins from round spermatids probed with an anti-ssH2B antiserum that recognizes the novel peptide sequence indicated that the protein was present in round spermatids but not in pachytene spermatocytes. The ssH2B comigrated with H2A.X by acid-urea (2.5 M) PAGE and migrated slightly more slowly than TH2B by acid-urea-Triton PAGE. When seminiferous tubules were microdissected stage-specifically and basic nuclear proteins were immunoblotted, ssH2B was detected in tubule sections of stages II-VI. It reached a maximum level in stages VII-VIII and then decreased to minimal levels in stages XIII-I. Since ssH2B was not detected in pachytene spermatocytes, the stage-specific levels of ssH2B corresponded to the respective steps of spermatids present in the tubules. While ssH2B constituted a relatively small amount (approximately 2%) of total H2B protein in round spermatids, its presence in both the mouse and the rat suggests that its function may be conserved in mammalian spermatogenic cells.

Animals↗
PubMed1999

Spermatid translocation in the rat seminiferous epithelium: coupling membrane trafficking machinery to a junction plaque.

In this study, we demonstrate that specialized junction plaques that occur between Sertoli cells and spermatids in the rat testis support microtubule translocation in vitro. During spermatogenesis, Sertoli cells are attached to spermatids by specialized adhesion junctions termed ectoplasmic specializations (ESs). These structures consist of regions of the plasma membrane adherent to the spermatid head, a submembrane layer of tightly packed actin filaments, and an attached cistern of endoplasmic reticulum. It has been proposed that motor proteins on the endoplasmic reticulum interact with adjacent microtubules to translocate the junction plaques, and hence the attached spermatids, within the epithelium. If this hypothesis is true, then isolated junctions should support microtubule transport. To verify this prediction, we have mechanically isolated rat spermatids, together with their attached ESs, and tested them for their ability to transport microtubules in vitro. Most assays were done in the presence of 2 mg/ml testicular cytosol and at room temperature. ESs attached to spermatids supported microtubule translocation. In some cases in which motility events were detected, microtubules moved smoothly over the junction site. In others, the movement was slow but progressive, saltatory and "inch-worm-like." No motility was detected in the absence of exogenous ATP or in the presence of apyrase (an enzyme that catalyses the breakdown of ATP). Our results are consistent with the microtubule-based motility hypothesis of spermatid translocation.

Animals↗
PubMed1999

A novel testicular haploid expressed gene (THEG) involved in mouse spermatid-sertoli cell interaction.

The successful production of male gametes requires extensive and precise interactions between germ cells and surrounding testicular somatic cells. We have made use of the mRNA differential display technique to isolate genes involved in germ cell-Sertoli cell interaction. We have identified five differential cDNA bands by comparing RNA from Sertoli cells, spermatids and spermatid-Sertoli cell cocultures. One of the isolated cDNA fragments detected a 1. 4-kilobase (kb) testis- and spermatid-specific transcript (designated as THEG: testicular haploid expressed gene). Northern blot analysis on RNA from spermatids and spermatid-Sertoli cell cocultures demonstrated that Sertoli cells are required for the continued expression of THEG in spermatids. We found two alternatively spliced transcripts for the THEG gene with 1437 base pairs (bp) and 1375 bp by using reverse transcription-polymerase chain reaction. The two open reading frames of 376 amino acids and 181 amino acids coded for putative nuclear proteins. The gene is approximately 10 kb pairs in size, contains 8 exons, and was mapped on mouse chromosome 10 to region B5-C1. Comparison of the two cDNA sequences with the genomic sequence indicated that the smaller transcript lacks exon 4. The differential gene expression of THEG in spermatid-Sertoli cell coculture supports the relevance of germ cell-Sertoli cell interaction for gene regulation during spermatogenesis.

Alternative Splicing↗
PubMed1989

Quantitative evaluation of the maintenance and development of spermatocytes and round spermatids in cultured tubule fragments from immature rat testis.

Maintenance and development of spermatocytes and round spermatids was studied in an in-vitro incubation system. This system consisted of open tubule fragments from 26-day-old rat testes, obtained after collagenase treatment. The tubule fragments contained Sertoli cells and spermatogenic cells up to and including a small number of early round spermatids. The number of primary spermatocytes and round spermatids in the tubule fragments was estimated using flow-cytometric analysis, immediately after isolation and after 72 h of incubation. In addition, the activity of LDH-C4 in the tubule fragments was measured. After 72 h of incubation, the percentage of spermatocytes was reduced by 70-80%, but the percentage of spermatids was doubled. The total LDH-C4 activity per well was increased 2-3-fold during 72 h of incubation of the fragments. A modest improvement of the culture results was observed when a combination of FSH, insulin, retinol and testosterone was added to the medium. LDH-C4 activity was investigated to see whether it could be used as a quantitative marker of isolated and cultured spermatocytes and spermatids. It was observed that LDH-C4 activity per cell was decreased when spermatocytes and spermatids were isolated and/or incubated at 4 degrees C. However, the cellular enzyme activity returned to control values during subsequent incubation of the cells at 32 degrees C, either in the absence or presence of a protein synthesis inhibitor. Cellular LDH-C4 activity may be influenced not only by temperature, but possibly also by other cell isolation conditions. It is concluded that LDH-C4 activity may not be a reliable quantitative marker for the presence of spermatocytes and spermatids in culture, but should be used in combination with other analytical methods such as DNA estimation and DNA flow cytometry.

Adenosine Triphosphate↗