Spermatogonial Divisions Research Articles

Germ cell transplantation as a tool for fertility preservation of oncological patients

Spermatogonia are the diploid cells in the testis, representing a small self-sustaining pool of male germ line stem cells as well as a larger pool of differentiating germ cells which enter the process of spermatogenesis. After going through prophase of meiosis as spermatocytes these cells finally leave the testis as sperm. Spermatogonia share many characteristics with other stem cells but also carry some specific physiological features. Many details about the kinetics of spermatogonial divisions, about the hormonal regulation of their expansion and differentiation and about their clinically relevant involvement as a cause for male infertility are poorly understood. However, the loss of spermatogonia following chemo- or radiotherapy leading to temporary or permanent infertility of the patient is a well known and unwanted site effect of many oncological therapies. Isolation and extracorporal storage of spermatogonia prior to the treatment and transplantation into the testis after recovery from the disease presents a promising future tool for fertility preservation in male patients undergoing oncological therapy.

Localization of two mammalian cyclin dependent kinases during mammalian meiosis.

Mammalian meiotic progression, like mitotic cell cycle progression, is regulated by cyclins and cyclin dependent kinases (CDKs). However, the unique requirements of meiosis (homologous synapsis, reciprocal recombination and the dual divisions that segregate first homologues, then sister chromatids) have led to different patterns of CDK expression. Here we show that Cdk4 colocalizes with replication protein A (RPA) on the synaptonemal complexes (SCs) of newly synapsed axes of homologously pairing bivalents, but disappears from these axes by mid-pachynema. The switch from the mitotic pattern of expression occurs during the last two spermatogonial divisions. Cdk2 colocalizes with MLH1, a mismatch repair protein at sites of reciprocal recombination in mid-late pachynema. In addition Cdk2 localizes to the telomeres of chromosomal bivalents throughout meiotic prophase. The mitotic pattern of expression of Cdk2 remains unchanged throughout the spermatogonial divisions, but is altered in meiosis of the spermatocytes.

Proteome analysis of spermatogonia: identification of a first set of 53 proteins

During spermatogenesis, the various classes of germ cells synthesize proteins necessary for their own functioning and for regulation of the Sertoli cells. However, the nature of these proteins has been little studied, especially in spermatogonia, the germ stem cells. In this study, the electrophoretic patterns of high-resolution, silver-stained, two-dimensional polyacrylamide gels of intracellular spermatogonial protein extracts were studied by computerized gel image analysis. We detected 675 individual spots, some of which we identified by mass spectrometry and database searching. We present here a first set of 53 proteins identified. They include housekeeping proteins never before detected in spermatogonia, ten proteins previously detected in the reproductive tract but not in spermatogonia, including stathmin, a protein previously shown to be involved in cell proliferation and differentiation, and one new testicular protein named translationally controlled tumor protein (TCTP), also known as a growth-related protein. Immunohistochemistry demonstrated that the two latter proteins were indeed highly expressed in spermatogonia in situ, and their possible involvement in spermatogonial division and proliferation is currently under investigation in our laboratory. We conclude that this type of experimental strategy, known as proteomics, is a very powerful way to analyze germ cell proteins comprehensively and should rapidly greatly improve our understanding of spermatogenesis.

Expression of mitotic cyclin B1 is not confined to proliferating cells in the rat testis.

Spermatogenesis is a precisely controlled and timed process comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, and the maturation and differentiation of haploid spermatids. Cell proliferation is controlled by genes involved in the regulation of the cell cycle. Among the principal regulatory proteins are cyclins, which are categorized according to their appearance during the cell cycle. B-type cyclins are mitotic cyclins and function at the G2/M transition of the cell cycle. We have investigated the expression and regulation of cyclin B1 during rat spermatogenesis. Rat cyclin B1 was isolated from a testis cDNA library and further used as a probe to detect mRNA expression. Northern blot hybridization of testis mRNA revealed the presence of a single 1.7-kilobase transcript. In situ hybridization showed stage-specific expression during spermatogenesis with highest expression found in late pachytene spermatocytes and early round spermatids. This pattern was confirmed in fractions of isolated germ cells. Immunocytochemistry displayed highest protein levels in round spermatids. Depletion of gonadotropins did not change the quantitative and qualitative expression pattern of cyclin B1. Therefore, the signals triggering the onset of cyclin B1 expression seem not to originate from the pituitary-gonadal endocrine axis and might therefore be paracrine factors originating within the germinal epithelium. Our observations suggest that cyclin B1 plays a hitherto unknown role in spermatid maturation in addition to its known function in dividing cells.

Fate of Germ Cells in 2,5-Hexanedione-Induced Testicular Injury: II. Atrophy Persists Due to a Reduced Stem Cell Mass and Ongoing Apoptosis

The Sertoli cell toxicant 2,5-hexanedione (2,5-HD) causes irreversible testicular atrophy in rats. After toxicant exposure, only Sertoli cells, stem cells, and a few spermatogonia remain in the seminiferous epithelium. In this study, the number, type, and fate of the remaining germ cells were determined. Male Sprague–Dawley rats were exposed to 1% 2,5-HD in drinking water for 5 weeks and then sacrificed 12 or 40 weeks after the start of exposure. Cell counts determined that the stem cell population was diminished in size, but made up a significant portion of the remaining germ cells. The remaining germ cells were primarily type A spermatogonia. Modeling of spermatogonial divisions suggested that most spermatogonia undergo degeneration at the level of type A3spermatogonia after 2,5-HD-induced atrophy. Apoptosis was demonstrated to occur in the remaining germ cells by nuclear morphology andin situanalysis of DNA fragmentation. Quantitation indicated that apoptosis occurred in a majority of stem cell progeny. We conclude that the irreversibility of 2,5-HD-induced testicular injury results from the reduced size of the stem cell population as well as a block in germ cell development at the level of type A spermatogonia.

Spermatogenesis in Tenebrio molitor (Tenebrionidae, Coleoptera): A Fine Structure and Anti‐tubulin Immunofluorescence Study

AbstractSpermatogonia and both generations of spermatocytes of Tenebrio molitor possess conventional bipolar spindles with only few aster MTs. Spindles in metaphase spermatogonia are surrounded by fenestrated two‐layered cisternae and do not contain intraspindle membranes. In metaphase spermatocytes, a spindle envelope is missing, but intraspindle membranes are abundant. Mitochondria form long threads lateral to the nucleus in prophase I of meiosis. The elongated mitochondria also align parallel to the spindle apparatus in prometaphase I. As a consequence, the spindles reside in a cage formed of mitochondria. This arrangement may guarantee proper bisection of the chondriome during division. Cells are tightly packed during spermatogonial divisions and in prophase I, but large intercellular spaces develop when the first meiotic spindle assembles. Then, cytoplasmic bridges which persist between the cells as a result of incomplete cytokinesis appear as slender tubes. Anti‐tubulin immunofluorescence using an antibody against acetylated α‐tubulin revealed intense acetylation throughout spermatogonial mitosis but a low degree of α‐tubulin acetylation in meiotic spindles prior to telophase. This may indicate a high microtubule turnover in meiosis.

Paracrystalline Endoplasmic Reticulum Is Typical of Gametogenesis in Hemiptera Species

The formations structure, and fate of paracrystalline endoplasmic reticulum (pcER) is described in male germ cells of the red firebug, Pyrrhocoris apterus (Pyrrhocoridae, Hemiptera, Insecta), using transmission electron microscopy of ultrathin sections. The pcER forms in spermatogonia close to cytoplasmic bridges interconnecting the cells. Compact aggregates of ER elements are visible there. The membrane masses appear very irregular and consist of anastomosing elements of agranular ER with an electron-dense lumen. Toward the end of spermatogonial divisions, the first signs of pcER formation are detectable in the form of circular and semicircular membrane profiles. In spermatocytes the pcER is fully developed. Most obvious in ultrathin sections through pcER blocks are circular, undulating, and almost straight membrane arrays. In advanced spermatids, pcER is missing. In order to determine whether pcER is widespread in Hemiptera, oocytes, spermatogonia, and spermatocytes of the cotton stainer, Dysdercus intermedius (Pyrrhocoridae), were also studied. Whereas oocytes and spermatogonia possessed pcER, it was absent from spermatocytes and spermatids. There were no hints to the function of the pcER, but the occurrence in germ cells of representatives of the Pyrrhocoridae renders them convenient systems for further experimental approaches.

The effect of theDrosophilaX chromosome on the number of male germ cells

Summary Fertile Drosophila subobscura males having the X chromosomal gene arrangement A2+3+5+7 produce predominantly female offspring. These males are called “sex ratio” males. Irrespective of the unisexual offspring, the total number of offspring of a sex ratio male can be as high as the bisexual offspring of control males. Thus the absolute number of female offspring is higher in A2+3+5+7 males than in control males. One reason for this high number of female offspring could be increased sperm production by sex ratio males. In the present study we analyzed spermatogenesis of sex ratio and control males. Sperm differentiation in Drosophila occurs in so-called cysts. In D. subobscura they contain, after spermatogonial divisions and meiosis, 128 synchronously developing spermatids each. We found that the testes of sex ratio males contained double the amount of cysts as control males. However, this is not the explanation for the great amount of female offspring of those males. In the final stage of spermatid maturation, in the coiling stage, the number of cysts was the same in sex ratio males as in control males. Because in sex ratio males only the X sperm are released into the seminal vesicle, these males transfer only half the number of functional sperm. The mechanism which is in charge of the high female offspring of sex ratio males could be a higher frequency of copulation and/or a better utilization of sperm by the female, or something else. The same high cyst number as in fertile A2+3+5+7 males was also found in sterile males with the same A chromosomal gene arrangement but different autosomal gene arrangements as fertile A2+3+5+7 males. This result indicates that the total number of cysts is controlled only by the A chromosome, independent of male fertility. In sterile A2+3+5+7 males sperm differentiation stopped during individualization. No sperm was found to enter the seminal vesicle.

Spermatogenic cells and stages of the seminiferous epithelial cycle in the Indian gerbil field rat, Tatera indica

Four types of spermatogonia (A0, A, In, B) are distinguished in T. indica. Four generations of A type, a single generation of intermediate (In) and two generations of B type spermatogonia are identified. Seven divisions of spermatogonia have been observed, in Stages 1b, 2b, 3b, 4b, 5, 6 and 7 respectively. Spermatocytes have been observed in prophase, metaphase, anaphase and telophase; in terms of size and morphology, the phases of prophase could be further divided. Pre-leptotene and leptotene appear in Stage 8 and Stage 1 respectively. Round spermatids become asymmetrical in Stage 1b. For the first time in rodents, Stages 1, 2, 3, 4 and 8 have been subdivided. The percentage frequencies of Stages 1-8 are 12.93, 10.69, 17.54, 10.33, 3.76, 11.20, 7.88 and 25.67 respectively. Although the morphology of the spermatogenic cells and associations of these cell types are basically similar to those in albino rats, T. indica markedly differs from all other rodents studied in having two generations of B spermatogonia and subdivisions of Stages 1, 2, 3, 4 and 8; in addition, the relative frequencies of the stages distinctly differ from those recorded in previous studies of rats. Hence, the durations of different cell types in T. indica may differ markedly from those in other rodents.

Structure, mitotic and meiotic behaviour, and stability of centromere-like elements devoid of chromosome arms in the fly Megaselia scalaris (Phoridae)

Minute elements detected in Megaselia scalaris (Phoridae, Diptera) lack chromosome arms but carry centromeres and possess kinetochore microtubules in mitosis as well as in meiosis. These centromere-like elements (CLEs) were present in two geographically independent strains of the fly. This indicates that their origin is not a recent event in the karyotype evolution of M. scalaris and that they are rather stable constituents of the karyotype. Most often, two CLEs were found in gonial and somatic mitosis. Spermatocytes contained one CLE. Two individuals examined deviated from this rule in that a metaphase spermatogonium showed three and an anaphase spermatogonium eight CLEs. These animals are believed to have been aneuploid relative to the CLEs. An analysis of spermatogonial division revealed that the CLEs behave like the centromeres of the regular chromosomes but seem to separate precociously, since they were closer to the spindle poles in late anaphase cells. Whereas the size of the CLEs was not significantly different between mitotic cells and secondary spermatocytes, the CLEs in primary spermatocytes were larger in volume by a factor of about 4.5 than those in mitosis and meiosis II. The additional material is interpreted as a glue that holds two CLEs together. This, in turn, is a prerequisite for orderly segregation. The function of the CLEs is not known. They are considered as B chromosomes reduced to the minimum required for segregation, the centromere.

Seasonal Differences in Equine Spermatocytogenesis1

Spermatocytogenesis plays a pivotal role in regulation of spermatogenesis; however, its details remain relatively obscure in nonrodent species. The equine testis contains approximately 100% more spermatogonia in summer than in winter and appears to be a good model to identify the flexible components of spermatocytogenesis that cause seasonal changes in daily sperm production. Testes were taken from horses in the winter (n = 47) and in summer (n = 43). Tissues were fixed by glutaraldehyde-perfusion and submission in osmium, embedded in Epon or methacrylate, sectioned at 0.5 micron or 5 microns, stained with toluidine blue, and observed using bright-field microscopy. The combined total number of A1, A2, A3, and B1 (A plus B1) spermatogonia/testis and the numbers of B2 spermatogonia or early primary spermatocytes were determined by stereology of Epon sections involving testicular volume density and volume of spermatogonial nuclei. In a subset of horses, different spermatogonial subtypes (A1, A2, A3, and B1) were counted per 100 Sertoli cells in each of the 8 spermatogenic stages and expressed as percentage of all A plus B1 spermatogonia. The number of each spermatogonial subtype/testis for the large series of horses was calculated by multiplying the number of A plus B1 spermatogonia/testis (determined for each horse) by the percentage of that given spermatogonial subtype. Season did not significantly affect the number of any given subtype per 100 Sertoli cells in any stage or percentages of different subtypes of spermatogonia. Numbers of A1 (p less than 0.05), A2, A3, B1, or B2 spermatogonia (p less than 0.01) were greater in the breeding season.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Insulin-Like Growth Factor I and Stage-Specific Levels of Epidermal Growth Factor in Stage Synchronized Rat Testes

Stage synchronization of seminiferous epithelium after withdrawal and replenishment of vitamin A provides a valuable and powerful approach to the investigation of paracrine interactions within the testis. However, since the discovery of this model, little attention has been given to the events surrounding the synchronous reinitiation of spermatogenesis after depletion of vitamin A. Synchronization of spermatogenesis was observed in all animals previously deficient in vitamin A. However, the degree of synchrony observed, as assessed by a ratio of synchrony, decreased markedly with time. The possibility that spermatogenic synchrony decreases with time due to variability of the temporal duration of stages of the cycle of the seminiferous epithelium is supported by this observation. However, long-term studies are required to substantiate this point. After initiation of stage synchrony of spermatogenesis, increased testicular concentrations of epidermal growth factor (EGF) were observed in testes synchronized between stages IX-II than at other stages of the cycle of the seminiferous epithelium. This elevation in testicular EGF concentrations correlated well with mitotic division of type A spermatogonia at stages IX, XII, and XIV of the cycle of the seminiferous epithelium. Previous in vitro studies have implicated an EGF-like factor in the stimulation of type A spermatogonial division in the mouse. A significant increase in testicular insulin-like growth factor I (IGF-I) concentrations was observed in control animals 14 days after the injection of retinol acetate. In vitamin A deficient animals, a marked increase in testicular IGF-I concentrations was observed as compared to age-matched controls. Maximal levels of testicular IGF-I concentrations were present 14 and 28 days and again 126 days after re-supplementation with retinol acetate. No stage dependent changes in testicular IGF-I were observed but the data provided suggest the retinol may be one of the factors involved in the regulation of testicular IGF-I.

Regulation of 'haploid expressed genes' in male germ cells

Department of Biology, Tufts University, Medford, MA 02155, USA Keywords: spermatogenesis; haploid gene expression; gene regulation; testis Introduction Spermatogenesis offers an experimental system whereby the gene expression of eukaryotic cells with tetraploid, diploid, and haploid chromosome complements can be compared. Starting from a population of stem cells, the diploid spermatogonia follow one of two lineages. One subpopulation of cells initiates a differentiation process ultimately leading to the spermatozoon while a second, presumably distinct, subpopulation of spermatogonia enters a pathway that maintains and repopulates the stem cells of the testis. The cells destined to become spermatozoa undergo several spermatogonial divisions. The last complete replication of DNA during spermatogenesis, in the preleptotene primary spermatocyte, heralds the start of meiosis. During the lengthy interval of meiotic prophase, homologous chromosomes synapse and genetic recombination occurs, producing the genetic diversity required for survival of a species. Following meiotic recombination, the 4N spermatocytes divide twice

Analysis of the testes of H-Y negative XOSxrb mice suggests that the spermatogenesis gene (Spy) acts during the differentiation of the A spermatogonia.

H-Y antigen negative XOSxrb mice, like their H-Y positive XOSxra counterparts, have testes; but, in contrast to XOSxra males, XOSxrb testes almost totally lack meiotic and postmeiotic stages of spermatogenesis. The quantitative analysis of the testes of XOSxrb males and their XY +/- Sxrb sibs, described in the present study, identified two distinct steps in this spermatogenic failure. First, there was a reduction in mitotic activity among T1 prospermatogonia, so that approximately half the normal number of T2 prospermatogonia were produced. Second, there was a dramatic decrease in the number of A3 and A4 spermatogonia and no Intermediate or B spermatogonia. These reductions were also largely due to decreased mitotic activity, there being a shortage of A1 and A2 spermatogonial divisions and no divisions among A3 or A4 spermatogonia. Mitotic activity among the T2 prospermatogonia and the undifferentiated A spermatogonia was normal. This means that the spermatogonial stem cells, which are a subset of the undifferentiated A spermatogonia, are unaffected in XOSxrb mice. Sxrb is now known to have arisen by deletion of DNA from Sxra. It is clear from the present findings that a gene (or genes) present in the deleted DNA plays a major role in the survival and proliferation of the differentiating A spermatogonia.

Intercellular bridges and factors determining their patterns in the grasshopper testis.

Intercellular bridges joining cells contained in cysts of Chortophaga viridifasciata testes were studied with light and electron microscopy. Preparations consisted of expressed whole cells (living, or fixed and stained) as well as sections. The secondary spermatogonia of each cyst are joined centrally by persisting fused interzonal bodies (fusomes) of incompletely cleaved cells. Shifts in cell orientation during anaphase are apparently responsible for central as opposed to chain linkage of cells. In the primary spermatocytes, the central fusome is replaced by a chain linkage, apparently resulting from the breakdown of the fusome into its original interzonal body components. Intercellular bridges are also present in spermatids, but there is no evidence to indicate the time of their formation (in the immediately preceding meiotic divisions or in the secondary spermatogonial divisions). The function of the compact centrally situated fusome in the secondary spermatogonial cyst is discussed as it relates to synchrony, number of cell divisions, spermatodesm formation, and fertility.

Gonadal structure and gametogenesis inScyllarus ardus(L.) (Crustacea, Decapoda)

Abstract The anatomy of male and female gonads, the oogenesis and the spermatogenesis of Scyllarus aretus are described. The different stages of meiosis are characterized by the nuclear behaviour and cell morphology. The storage of fully vitellinized oocytes and the amount of oogonia were observed throughout the year. The relationship between oogonial multiplication and development of the oocytes is described. The development of the spermatogonial ridges was studied; their frequency showed remarkable variations during the year: the periods of maximal spermatogonial division preceded those of maximal amounts of fully vitellinized oocytes. It was possible, therefore, to correlate male production of meiocytes with egg production and release.

Seasonal Changes in Spermatogenesis (Including Germ Cell Degeneration) and Plasma Testosterone Concentration in the Grey-headed Fruit Bat, Pteropus poliocephalus

Maximal testicular size of P. poliocephalus occurred during the February-March-April period (autumn breeding season) associated with maximal plasma levels of testosterone (121' 5 � 23� 4 nmol/I). Testicular size decreased after the breeding season concomitant with a decrease in plasma testosterone (7'6 � 1�7 nmol/I). Histologically, regressive changes were observed in the testis after the breeding season including a decrease in seminiferous tubule diameter and a decrease in Leydig cell nuclear diameter. Significant germ cell loss occurred during the breeding season (44%) mainly during the final spermatogonial division and meiosis and increased in regressed testes (69%), being accentuated chiefly at meiosis. All regressed testes showed some sperm production although it was much reduced after the breeding season; however, optimal fertility appears to be restricted to autumn by the large increases in testosterone secretion which only occur at this time.

Ultrastructure of the kinetochore inGraphosoma italicum (Hemiptera: Heteroptera)

Ultrastructural analysis of the centromere in the germ-line ofGraphosoma italicum (Hemiptera: Heteroptera) (2n=12 + XY♂) revealed differences between mitotic and meiotic chromosomes. In mitotic spermatogonial divisions a trilaminar kinetochore plate extending almost the entire length of the chromosomes is present. Meiotic chromosomes, on the contrary, lack trilamellar kinetochore plate, although one chromosome end exhibits a round structure which is denser than the remainder chromatin and resembles a “ball and cup” kinetochore. These structures are orientated towards the poles and sometimes show associated microtubules. Additionally, the meiotic chromosomes are surrounded by a complex system of membranes. The possible role of the round structure and of the complex membrane system in meiotic segregation is discussed.

Inhibition of canine spermatogonial mitosis with follicle regulatory protein.

Evidence was found that the Graafian follicle produces and secretes a hormone-like protein, referred to as follicle regulatory protein (FRP), which affects the responsivity of other follicles to gonadotropin stimulation. Since a similar material was identified in homogenates of bull testes, we evaluated the effects of FRP on the histology of seminiferous epithelium by its systemic injection into normal dogs. Mongrel dogs were injected IM with 5 mg of FRP for 17 consecutive days. The seminiferous epithelium was evaluated by light microscopy and divided into eight stages according to Foote. The relative frequency of stage 4 was significantly less in the FRP-treated than in the control group, but that of stage 7 was significantly greater in the FRP-treated group. The ratios of type B/type A spermatogonia for control and FRP-treated groups for stages 6-8 were 1.5, 5.8, 2.4, and 1.6, 4.3, 1.6, respectively. The tubular diameters of stages 6 and 7 were significantly different between the two groups. The numbers of tubules containing spermatogonial divisions were significantly decreased at stages 2 + 3 and stages 6 + 7 in the FRP-treated group. A clear decrease in the number of tubule profiles with spermatogonial divisions was present at stages 6 and 7 in the FRP-treated testis. Taken together, these findings suggest that FRP inhibits spermatogonial transformation of type A into type B.

Seasonal variation in semen quality of bulls and correlations with metabolic and endocrine parameters.

Sperm output and semen quality of 17 bulls sampled over 12 months showed minimal output in mid-winter and late summer and minimum quality in late summer. Monthly measurements of luteinising hormone and testosterone concentration in plasma and testosterone concentration in semen were made over 12 months. Serum non-esterified fatty acids (NEFA), albumin and total protein were also measured for the final seven months of this period. Plasma testosterone showed a strong negative correlation with sperm numbers two months hence but not in the current month. Plasma testosterone by bull and semen testosterone by month was also correlated with sperm output. Plasma luteinising hormone three months and serum total protein two months prior was positively correlated with sperm numbers ejaculated and the normality of sperm morphology, possibly by affecting the luteinising hormone dependent A0 to A1 spermatogonial division. NEFA was correlated with initial and post freezing/thawing motility in the current month, possibly by affecting membrane stability. The value of examining the bull in diagnosing infertility of cows where nutritional stress may have occurred is suggested, as is the use of albumin/total protein and NEFA measurements as a prognostic aid for time to return to normality of function of such bulls.