Late Primary Spermatocytes Research Articles

Characterization of sheep spermatogenesis through single-cell RNA sequencing.

Spermatogenesis is an important biological process in male reproduction. The interaction between male germ cells and somatic cells during spermatogenesis, is necessary for male reproductive activities. This cellular heterogeneity has made it difficult to profile distinct cell types at different stages of development. Here, we present the first comprehensive, unbiased single-cell transcriptomic study of sheep spermatogenesis using 10× genomics single cell sequencing (scRNA-seq). We collected scRNA-seq data from 11772 cells from the adult sheep testis and identified all known germ cells (including early primary spermatocytes, late primary spermatocytes, round spermatids, elongated spermatids, and sperm), and somatic cells (Sertoli cells and Leydig cells), as well as one somatic cell that unexpectedly contained leukocytes. The functional enrichment analysis indicated that several pathways of cell cycle, gamete generation, protein processing, and mRNA surveillance pathways were significantly enriched in testicular germ cell types, and ribosome pathway was significantly enriched in testicular somatic cell types. Further analysis identified several stage-specific marker genes of sheep germ cells, such as EZH2, SOX18, SCP2, PCNA, and PRKCD. Our research explored for the first time of the changes in the transcription level of various cell types during the process of sheep spermatogenesis, providing new insights for sheep spermatogenesis and spermatogenic cell development.

Post-meiotic transcription inDrosophilatestes

Post-meiotic transcription was accepted to be essentially absent from Drosophila spermatogenesis. We identify 24 Drosophila genes whose mRNAs are most abundant in elongating spermatids. By single-cyst quantitative RT-PCR, we demonstrate post-meiotic transcription of these genes. We conclude that transcription stops in Drosophila late primary spermatocytes, then is reactivated by two pathways for a few loci just before histone-to-transition protein-to-protamine chromatin remodelling in spermiogenesis. These mRNAs localise to a small region at the distal elongating end of the spermatid bundles, thus they represent a new class of sub-cellularly localised mRNAs. Mutants for a post-meiotically transcribed gene (scotti), are male sterile, and show spermatid individualisation defects, indicating a function in late spermiogenesis.

A quantitative (stereological) study of the effects of experimental unilateral cryptorchidism and subsequent orchiopexy on spermatogenesis in adult rabbit testis

The aim of this study was to examine the controversial effects of experimental unilateral cryptorchidism and subsequent orchiopexy on the number of germ cells and other morphometric characteristics of testicular and epididymal structures in adult rabbits. Unilateral cryptorchidism was induced in 11 mature male New Zealand white rabbits by returning one testis, together with the ipsilateral epididymis, to the abdominal cavity via a surgical procedure. After 3 months, testes and epididymides were removed from six animals (and from six age-matched control animals that did not undergo the surgery). Orchiopexy was performed on the five remaining animals and the testes and epididymides of these animals (and an additional six age-matched control animals) were removed 7 weeks later. A contemporary, unbiased and efficient stereological tool, the optical disector, was used to estimate the number of nuclei in the testis and epididymis using methacrylate-embedded sections of 25 micron in thickness. Cryptorchidism resulted in severe testicular atrophy and spermatogenic arrest: type A spermatogonia and Sertoli cells only were seen in the seminiferous epithelium, and the number of type A spermatogonia per testis was reduced by 84%. After orchiopexy, the testis remained atrophied and the number of type A spermatogonia returned to the near-normal range in four of five animals, but spermatogenesis was recovered only partially at the stage of early primary spermatocytes (one animal), late primary spermatocytes (two animals) or spermatids (one animal). In conclusion, cryptorchidism caused severe spermatogenic arrest that was potentially recoverable (in view of the restoration of the number of type A spermatogonia), but orchiopexy failed to induce full recovery of spermatogenesis.

A quantitative (stereological) study of the effects of experimental unilateral cryptorchidism and subsequent orchiopexy on spermatogenesis in adult rabbit testis.

The aim of this study was to examine the controversial effects of experimental unilateral cryptorchidism and subsequent orchiopexy on the number of germ cells and other morphometric characteristics of testicular and epididymal structures in adult rabbits. Unilateral cryptorchidism was induced in 11 mature male New Zealand white rabbits by returning one testis, together with the ipsilateral epididymis, to the abdominal cavity via a surgical procedure. After 3 months, testes and epididymides were removed from six animals (and from six age-matched control animals that did not undergo the surgery). Orchiopexy was performed on the five remaining animals and the testes and epididymides of these animals (and an additional six age-matched control animals) were removed 7 weeks later. A contemporary, unbiased and efficient stereological tool, the optical disector, was used to estimate the number of nuclei in the testis and epididymis using methacrylate-embedded sections of 25 micron in thickness. Cryptorchidism resulted in severe testicular atrophy and spermatogenic arrest: type A spermatogonia and Sertoli cells only were seen in the seminiferous epithelium, and the number of type A spermatogonia per testis was reduced by 84%. After orchiopexy, the testis remained atrophied and the number of type A spermatogonia returned to the near-normal range in four of five animals, but spermatogenesis was recovered only partially at the stage of early primary spermatocytes (one animal), late primary spermatocytes (two animals) or spermatids (one animal). In conclusion, cryptorchidism caused severe spermatogenic arrest that was potentially recoverable (in view of the restoration of the number of type A spermatogonia), but orchiopexy failed to induce full recovery of spermatogenesis.

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 ± 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 ± 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.

Stage-specific expression of B cell translocation gene 1 in rat testis

B Cell translocation gene 1 (BTG1) is a member of a new family of putative antiproliferative factors. They are characterized by their rapid, but transient, expression in response to factors that induce growth arrest and subsequent differentiation. In immature rat Sertoli cell cultures, BTG1 messenger RNA (mRNA) increases rapidly after FSH stimulation. We obtained the full-length coding sequence of rat BTG1 complementary DNA for Northern blot analysis and in situ hybridization to determine the temporal expression and spatial distribution of BTG1 mRNA in the rat testis. Northern analysis of isolated adult germ cells and in situ hybridization analysis of adult seminiferous epithelium demonstrated that BTG1 expression was first evident in late primary spermatocytes. The level of BTG1 mRNA was also elevated in secondary spermatocytes, but was maximal in postmeiotic round spermatids where levels were 5 times the background. BTG1 mRNA was not detectable in cells in the M phase of meiosis or spermatids undergoing nuclear elongation and condensation. The oscillation of BTG1 expression from the late prophase of the first meiotic division through spermatozoa release suggests BTG1 involvement in spermatogenesis. High levels of BTG1 mRNA at entry into terminal spermatid differentiation suggests a role consistent with that proposed for the BTG1 family of antiproliferative factors.

Stage-specific expression of B cell translocation gene 1 in rat testis.

B Cell translocation gene 1 (BTG1) is a member of a new family of putative antiproliferative factors. They are characterized by their rapid, but transient, expression in response to factors that induce growth arrest and subsequent differentiation. In immature rat Sertoli cell cultures, BTG1 messenger RNA (mRNA) increases rapidly after FSH stimulation. We obtained the full-length coding sequence of rat BTG1 complementary DNA for Northern blot analysis and in situ hybridization to determine the temporal expression and spatial distribution of BTG1 mRNA in the rat testis. Northern analysis of isolated adult germ cells and in situ hybridization analysis of adult seminiferous epithelium demonstrated that BTG1 expression was first evident in late primary spermatocytes. The level of BTG1 mRNA was also elevated in secondary spermatocytes, but was maximal in postmeiotic round spermatids where levels were 5 times the background. BTG1 mRNA was not detectable in cells in the M phase of meiosis or spermatids undergoing nuclear elongation and condensation. The oscillation of BTG1 expression from the late prophase of the first meiotic division through spermatozoa release suggests BTG1 involvement in spermatogenesis. High levels of BTG1 mRNA at entry into terminal spermatid differentiation suggests a role consistent with that proposed for the BTG1 family of antiproliferative factors.

Two different patterns of interrupted spermatogenesis in winter diapause and summer quiescence in the desert beetle, Omorgus freyi.

Spermatogenesis discontinues during both adult reproductive diapause and quiescence in the flightless males of Omorgus freyi. During both types of dormancy, spermatogenesis discontinuity is a dynamic process involving an uninterrupted supply of primary spermatocytes that undergoes partial development and lyses before spermatozoa can be formed. Notwithstanding this common feature, the pattern of discontinuity differs between the two kinds of dormancy. During reproductive diapause, spermatozoa and late spermatids are the first cells that lyse; after diapause break, spermatozoa are produced anew. During quiescence, in contrast, the first indication of discontinuity is the degeneration of late primary spermatocytes; spermatozoa apparently remain intact for a while but eventually degenerate as well. Therefore, males returning to sexual activity after a short period of quiescence may still have spermatozoa capable of fertilization. However, following a long period of quiescence, spermatozoa will be absent and must be produced anew, as in postdiapausing males. The existence of two different patterns of spermatogenesis discontinuity may indicate that each is controlled differently by the endocrine system, in response to the different environmental conditions inducing quiescence or diapause. Dual control of discontinuity may contribute to the high reproductive potential of these flightless beetles, under unpredictable and extreme desert conditions. © 1993 Wiley-Liss, Inc.

Expression of inhibin beta A and beta B, follistatin and activin-A receptor messenger ribonucleic acids in the rat seminiferous epithelium.

The expression of inhibin beta A and beta B subunits, follistatin, and activin-A receptor messenger RNA (mRNAs) in different stages of rat seminiferous epithelial cycle was analyzed by in situ hybridization in order to understand their role in the regulation of spermatogenesis. Inhibin beta A mRNA was expressed in Sertoli cells in a highly stage-specific manner. The mRNA levels started to accumulate in Sertoli cells at stage VIII of the cycle and were highly expressed during stages IX-XI. Follistatin mRNA expression was identical to that of inhibin beta A, while inhibin beta B mRNA was maximally expressed in Sertoli cells at stages XIII-III. Low expression was found in stages VII-VIII. Activin-A receptor mRNA was localized mainly in spermatogenic cells. Maximal expression was seen in late primary spermatocytes at stages XIII-XIV and in early round spermatids at stages I-IV. A low even expression by Sertoli cells was also seen. Inhibin beta A and follistatin mRNAs were coexpressed in stage IX-XI Sertoli cells, suggesting close interplay between these molecules. The pattern of inhibin beta B mRNA expression was similar to that of inhibin alpha-mRNA. Localization of activin-A receptor mRNA in spermatogenic cells suggests that activin may influence meiotic divisions and early spermiogenesis.

The effects of capture stress on testis function in the Australian Swamp buffalo ( [formula omitted][formula omitted

Capture stress significantly affected the cellular composition and function of the testis and epididymis of feral Swamp buffalo bulls. There was an initial acute drop in the population of elongated spermatids, round spermatids and late primary spermatocytes, and subsequently, a more gradual decline in the early elongated spermatid and late primary spermatocyte populations as the duration of the stress increased. Sertoli cell numbers were unaffected. The most marked effect was on the early elongated spermatid population, which was approximately halved in the stressed bulls. Testis parencyhmal weight was approximately 50% lower in stressed bulls. Affected bulls would undoubtedly be at least temporarily subfertile or infertile. The long-term prognosis on the fertility of such bulls is not known. Possible mechanisms involved in the pathogenesis of the syndrome are considered in this study.

Genomic organization of major sperm protein genes and pseudogenes in the nematode Caenorhabditis elegans

The major sperm proteins (MSPs) are a family of closely related, small, basic proteins comprising 15% of the protein in Caenorhabditis elegans sperm. They are encoded by a multigene family of more than 50 genes, including many pseudogenes. MSP gene transcription occurs only in late primary spermatocytes. In order to study the genomic organization of transcribed MSP genes, probes specific for the 3′ untranslated regions of sequenced cDNA clones were used to isolate transcribed genes from genomic libraries. These and other clones of MSP genes were located in overlapping cosmid clones by DNA fingerprinting. These cosmids were aligned with the genetic map by overlap with known genes or in-situ hybridization to chromosomes. Of 40 MSP genes identified, 37, including all those known to be transcribed, are organized into six clusters composed of 3 to 13 genes each. Within each cluster, MSP genes are not in tandem but are separated by at least several thousand bases of DNA. Pseudogenes are interspersed among functional genes. Genes with similar 3′ untranslated sequences are in the same cluster. The six MSP clusters are confined to only three chromosomal loci; one on the left arm of chromosome II and two near the middle of chromosome IV. Additional sperm-specific genes are located in one cluster of MSP genes on chromosome IV. The multiplicity of MSP genes appears to be a mechanism for enhancing MSP synthesis in spermatocytes, and the loose clustering of genes could be a result of the mechanism of gene duplication or could play a role in regulation.

Quantification of human spermatogenesis: germ cell degeneration during spermatocytogenesis and meiosis in testes from younger and older adult men.

Germ cell degeneration during spermatocytogenesis and meiosis was investigated to explain the age-related decline in daily sperm production (DSP). Numbers of Types A-dark, A-pale, and B-spermatogonia, potential daily sperm production per g parenchyma (PDSP) based on type B-spermatogonia, early primary spermatocytes, and late primary spermatocytes, and DSP per g based on early spermatids were determined in 15 men aged 20 to 48 yr (mean +/- SEM, 33 +/- 2 yr) and 15 men aged 52 to 90 yr (65 +/- 3 yr). Testes obtained within 15 h of death (largely due to trauma or heart failure) were perfused vascularly with glutaraldehyde. The number of each cell type per g parenchyma was calculated as the product of the percentage of nuclei in the parenchyma times a correction factor for section thickness and nuclear diameter divided by the volume of a single nucleus of that cell type. Paired testicular weight was lower (p less than 0.01) in older men (33 +/- 3 g) than in the younger men (49 +/- 3 g). Younger and older men had similar numbers of A-dark, A-pale, and B-spermatogonia per g parenchyma. PDSP based on late primary spermatocytes and DSP based on early spermatids were lower (p less than 0.01) in older men than in younger men. In younger men, PDSP was similar (p greater than 0.05) between B-spermatogonia and late primary spermatocytes, whereas DSP measured at the spermatid level was abruptly lower than that estimated from younger cell types. Older men showed reduction in PDSP between early and late primary spermatocytes, with further reduction occurring in DSP at the spermatid level.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium and potassium currents in spermatogenic cells dissociated from rat seminiferous tubules.

The electrophysiological properties of the cell membrane of rat spermatogenic cells were studied using the whole-cell variation of the patch-electrode voltage-clamp technique. In late primary spermatocytes and early spermatids isolated from adult testis, a transient inward current followed by a slowly developing outward current was produced when the membrane potential was made more positive than -60 mV. Early spermatogenic cells which consist of spermatogonia and early spermatocytes were isolated either from new-born rats (12-14 days old) of from adult cryptorchid rats 15-21 days after the operation. In early spermatogenic cells, some showed a slowly developing outward current with negligible initial inward current, while others showed a recognizable inward current followed by the slowly developing outward current. The inward currents are identified as Ca2+-carried current, since replacement of external Ca2+ with Mn2+ reversibly diminished the current whereas Ba2+ or Sr2+ substituted for Ca2+. The reversal potential of the outward current changed from -65 to -12 mV when [K+]o was raised from 5 to 100 mM. The outward current was independent of [Ca2+]o and was blocked by tetraethylammonium chloride. Thus the current was identified as membrane-potential-dependent K+ current. During spermatogenesis from spermatogonia to early spermatids, the density of Ca2+ current increased while the K+ current density decreased significantly.

The effect of acute far field exposure at 2.45 GHz on the mouse testis.

Male C3H mice were exposed in an anechoic chamber to 2.45 GHz microwave radiation. The exposures ranged from 1000 Wm-2 for 5 min to 100 Wm-2 for 260 min, giving dose rates to the testis ranging from 66 Wkg-1 to 7 Wkg-1. The mice were killed six days later and the testes examined histologically. Quantitatively, no significant effects were seen on cells identified as X-ray sensitive (spermatogonia type B) or heat sensitive (early primary spermatocytes and late primary and secondary spermatocytes) or on the sperm count. Extrapolation of these results to man would suggest that acute exposure to the Maximum Permissible Exposure level of 100 Wm-2 has no effect on the testes in the ranges 1 to 3.5 MHz and 300 MHz to 100 GHz. There was insufficient data to comment on other frequencies.

Studies on H-Y antigen in different cell fractions of the testis during pubescence: immature germ cells are H-Y antigen negative.

Various cell types of the rat testis during pubescence, including germ, Sertoli, and Leydig cells, were partially enriched. The fractions were tested for the presence, binding, and secretion of H-Y antigen. The main results are: Immature germ cells are H-Y antigen-negative until the late diploid stages, and late primary spermatocytes or spermatids become positive; the somatic cells of the gonad are positive at all ages examined (18 days old to adulthood). Secretion of H-Y antigen is restricted to the Sertoli cell fraction. Binding of externally supplied antigen takes place on Leydig cells; the Sertoli cell surface will be saturated because of active secretion; there is no binding to germ cells. Thus, immature germ cells seem to be the only H-Y antigen-negative cells of the male organism, and the Sertoli cells seem to be the only ones to secrete H-Y antigen.

Cytoplasmic inclusions in Xenopus spermatogenic cells. ultrastructural and cytochemical analysis of the action of antimitotic agents on subcellular elements

ABSTRACT The effects of vinblastine sulphate, alone and in combination with other antimitotic agents, were tested in vitro on testicular tissue from the anuran, Xenopus laevis. Treatment with vinblastine alone resulted in dissolution of spindle and intercellular bridge microtubules, but centrioles and sperm tail axonemes remained intact. In addition, no paracrystalloid inclusions typical of those reported in other cell types treated with vinblastine alone were observed in Xenopus spermatogenic cells. Such inclusions were formed, however, when vinblastine was administered in combination with colchicine, colcemid, or puromycin. Under optimal conditions, paracrystalloids were formed in all cells from primary spermatogonia to late spermatids. The maximum size paracrystalloid formed differed in different spermatogenic cell stages, with late primary spermatocytes containing the largest such inclusions, often averaging over 34 μ m3 each. Autoradiographic analysis of spermatogenic cells labelled with [3H]leucine prior to drug treatment indicated that some radioactive material was incorporated into paracrystalloids in all cell stages. Treatment of Xenopus spermatogenic cells with vinblastine and puromycin was also employed to investigate the possible relationship between paracrystalloid material and a naturally occurring spermatogenic cell inclusion, the chromatoid body, since previous experiments indicated that microtubule protein might contribute to the mass of this structure. It was found that under certain conditions, both paracrystalloids and giant chromatoid bodies can occur together in the same cell, each inclusion reaching a characteristic size independent of the other. This suggests that the two inclusions are aggregates of materials drawn from different subcellular precursor pools. In addition, autoradiographic analysis of isotope incorporation indicated that giant chromatoid bodies could be clearly labelled with tritiated amino acids, but not with [3H]uridine. The pattern of isotope incorporation observed in giant chromatoid bodies, coupled with their histochemical staining characteristics, rules out the possibility that these structures function as cytoplasmic nucleoli.