Mitotic instability leading to an accumulation of B-chromosomes in grasshoppers

The study of mitotically unstable B-chromosomes (supernumeraries) of two grasshopper species confirmed a suggestion made earlier (Nur, 1963) that the instability should always be associated with a tendency of the B's to increase in frequency. Among 780 Camnula pellucida (Scudder) males from California, 105 had B's. In the testes of these males the number of B's varied from follicle to follicle and ranged between 0 and 4. Because of this variation, the number with which each male started to develop could not be determined. However, the relatively low frequency of males with B's and the regular meiotic behavior of the latter suggested that most of the 105 males started with a single B. Cytological analysis of the cells of the gastric caeca of 31 males whose testes contained B's confirmed this suggestion by showing that only one male had two B's in these cells; all the rest had one. In the testes of the 74 other males the mean number of B's ranged from 0.89–2.50, but only two males had means higher than 2.00. The observed ratio of one male with two B's to 30 with one, suggested that only the two males with the highest means started to develop with two B's and that the other 72 males all started with one. Since the mean for the 72 males was 1.37 B's per male, it was concluded that during the development of the testes of these males the mean increased by 37%. The males with B's had fewer follicles in their testes and apparently had also a lower frequency of normal sperm. — The analysis of the testes of Locusta migratoria L. males from Japan gave results which agreed with those from C. pellucida.Supported by grants GB 1585 and GB 6745 from the National Science Foundation, Washington, D.C.     

Morphology of the X-irradiated testes of Dermestes frischii Kugelann (Coleoptera: Dermestidae). I. Somatic cells and pre-spermatid germ cells

During the development of a sterile male control method for Dermestes frischii Kugelann, testis follicles exposed to an X-ray dose of 3.0 krad were investigated using light and electron microscopy.

There was considerable variation in the radiation sensitivity of the various somatic and germ cells. The cyst wall cells seemed particularly resistant while the inner layer of the bilayered follicle sheath was destroyed. The general resistance and versatility of the outer sheath layer maintained the integrity of the follicles. The only outer sheath and apical cells observed to decay were those in close proximity to degenerating germ cells. In some follicles all primary spermatogonia were destroyed, in others their numbers were only depleted. The surviving cells all underwent mitotic delay for 7–14 days. Their subsequent offspring were frequently found to break down and sometimes were proliferated so that germarial polarity was lost. All secondary spermatogonia but only a few primary spermatocytes were destroyed. The decay of germ cells within the same cyst did not necessarily proceed synchronously.     

The B-chromosomes of Locusta migratoria I. Detection of negative correlation between mean chiasma frequency and the rate of accumulation of the B's; a reanalysis of the available data about the transmission of these B-chromosomes

B-chromosomes were studied in two Spanish populations of Locusta migratoria. Both exhibit a high frequency of B-carrying individuals (75% and 94%). In both the B-chromosomes are mitotically unstable and they accumulate in the male germ line. The mean rate of accumulation is 28.5% in Baños de la Encina and 31.9% in Carboneras. There are no significant differences in mean cell chiasma frequency and between-cell variance of chiasmata between follicles with different number of B's within individual males. Likewise, there are no significant differences in mean cell chiasma frequency and between-cell variance between males with and without supernumeraries. However, a significant negative correlation exists between mean chiasma frequency and the rate of accumulation.     

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.     

Germ cells in Hydra oligactis males. I. Isolation of a subpopulation of interstitial cells that is developmentally restricted to sperm production

Single clones of interstitial cells were generated and analyzed to determine if one interstitial cell has the capacity to differentiate both somatic and germ cells. Such clones were produced by using hydroxyurea to selectively eliminate interstitial cells from normal Hydra oligactis males. The number of animals devoid of interstitial cells within the population was determined by staining whole animals with toluidine blue which renders the interstitial cells visible. The number of animals containing single clones of interstitial cells was then estimated using single hit Poisson statistics. In treatments which rendered 60-80% of the population devoid of interstitial cells, the majority of the animals containing interstitial cells lost the ability to produce somatic cells, including nerves and nematocytes, but retained the capacity to produce sperm. This result strongly suggests the presence of a separate germ line in hydra.     

Differentiation of Primary Spermatocytes to Elongated Spermatids by Mammalian FSH in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster

Our previous studies (10, 11) showed that mammalian follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the initiation and promotion of spermatogenesis from secondary spermatogonia to primary spermatocytes in organ culture of testes fragments from the newt, Cynops pyrrhogaster. The present study demonstrated that FSH promoted in the same model system the differentiation of primary spermatocytes even further: to the stage of elongated spermatids. When testes fragments, consisting of somatic cells and germ cells (mostly primary spermatocytes), were cultured in a control medium for three weeks, only round spermatids and spermatogonia were observed; both the diameter of the cysts and the viability of the germ cells decreased to about 10–15% of the original level. On the other hand, when the medium was supplemented with FSH, elongated spermatids appeared by the second week; both the diameter of the cysts and the viability of the germ cells were maintained at a higher level than in the control medium. The effect of FSH was dose-dependent. However, neither transferrin, androgens (testosterone and 5α-dihydrotestosterone) nor luteinizing hormone (LH) was effective. The addition of cyanoketone, a specific inhibitor of 3β-hydroxy-Δ⁵-steroid dehydrogenase (3β-HSD) (32), to the FSH-containing medium did not prevent the differentiation promoted by FSH, indicating that it is unlikely that Δ⁴-steroid metabolites produced in fragments by FSH acted directly on germ cells. Insulin was found to improve the viability of germ cells during a 2 week of culture period. In the presence of FSH, the cells in various differentiative stages had morphological characteristics very similar to those in vivo, whereas in the absence of FSH primary spermatocytes showed abnormal features in their nuclei and cytoplasm, indicating that they were deteriorating. These results and our previous results (1–3) suggest that FSH promotes primary spermatocytes to differentiate into elongated spermatids probably by stimulating Sertoli cells to secrete factors which then act on the germ cells.     

Centrosome inheritance in the male germ line of Drosophila requires hu-li tai-shao function

Cytokinesis partitions a centrosome to each daughter cell at cell division that will duplicate and assemble a bipolar spindle in the subsequent M phase. Cytokinesis is incomplete in proliferating germ cells in Drosophila and cytoplasmic channels connect sibling germ cells. Although centrosomes are essential to male fertility, the molecular mechanism that retains centrosomes in parental germ cells is not known. Cortical cytoplasmic structures known as fusomes extend through ring canals and connect cells within the cyst. Fusome assembly in males requires function of hu-li tai-shao (hts), an adducin like protein found in fusomes and in the cortical membrane cytoskeleton of somatic cells. This work used immunological and cytological methods to place hts mutants in an allelic series. Male fertile hts mutants express hts protein and generate apparently normal or fragmented fusomes. A male sterile allele does not express hts protein or show fusome structures. Gonial cells in all hts mutants showed 2 centrosomes and mitotic spindles were bipolar. Yet, primary spermatocytes, with and without fusome structures, frequently contained too many or too few centrosomes. Although spindle structures were not found in spermatocytes without centrosomes, meiotic spermatocytes with centrosomes generated bipolar, monopolar, and multipolar spindles. Collectively, these results indicate that hts function is necessary for centrosome inheritance in spermatocytes as well as for male fertility.     

IGSF11 is required for pericentric heterochromatin dissociation during meiotic diplotene

Meiosis initiation and progression are regulated by both germ cells and gonadal somatic cells. However, little is known about what genes or proteins connecting somatic and germ cells are required for this regulation. Our results show that deficiency for adhesion molecule IGSF11, which is expressed in both Sertoli cells and germ cells, leads to male infertility in mice. Combining a new meiotic fluorescent reporter system with testicular cell transplantation, we demonstrated that IGSF11 is required in both somatic cells and spermatogenic cells for primary spermatocyte development. In the absence of IGSF11, spermatocytes proceed through pachytene, but the pericentric heterochromatin of nonhomologous chromosomes remains inappropriately clustered from late pachytene onward, resulting in undissolved interchromosomal interactions. Hi-C analysis reveals elevated levels of interchromosomal interactions occurring mostly at the chromosome ends. Collectively, our data elucidates that IGSF11 in somatic cells and germ cells is required for pericentric heterochromatin dissociation during diplotene in mouse primary spermatocytes.     

Replication of euchromatin and heterochromatin in a mealybug

Asynchronous DNA replication of euchromatic (E) and heterochromatic (H) chromosomes and heterochromatic B chromosomes (B) were studied in the mealybug, Pseudococcus obscurus Essig (Homoptera: Coccoidea). The study was carried out on mycetocytes of adult females and on spermatocytes of mid-second instar males by employing tritiated thymidine labeling and autoradiography. In the mycetocytes the incorporation of the labeled thymidine began and ended later in the B's than in the E chromosomes. The S period was found to be about 21 hours. The DNA replication of the E chromosomes occupied about 86% of the S period and that of the B's 33%; during 18% of the mid-S period the replication of the two types of chromosomes overlapped. In the meiotic S period of the spermatocytes, the DNA of the E chromosomes started to replicate earlier than that of the H chromosomes and the B's, but the replication of the E chromosomes, the H chromosomes, and the B's overlapped. The H chromosomes completed their replication much later than the E chromosomes and slightly later than the B's.Supported by grants GB 1585 and GB 6745 to Dr. Uzi Nur from the National Science Foundation, Washington, D. C.Part of a thesis submitted to the University of Rochester in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Follicle cells and germ line cells both affect polarity in dicephalic chimeric follicles of Drosophila

Summary In aberrant egg follicles of the pattern mutant dicephalic (dic) the oocyte is wedged in between two groups of nurse cells, and this condition may give rise to embryos which express anterior traits at both ends. We have analysed the role of the dic genotype of the germ line cells and the surrounding somatic follicle cells in the formation of the dic follicular phenotype. By means of pole cell transplantations into Fs (1) K 1237 hosts (this cell-autonomous mutation causes degeneration of the host's germ line cells early in oogenesis), we constructed chimeras in which either the follicle cells, the germ line cells, or both were homozygous for the dic mutation. In all three combinations the dic phenotype was expressed but not in controls with dic ⁺ in both germ line cells and follicular epithelium. Since follicles with the dic phenotype may be produced if either the germ line cells or the follicle cells lack dic ⁺ gene activity we suggest that cellular interactions between both cell types are required for the correct positioning of the oocyte at the follicle's posterior pole.     

Evidence against a (2)n synchronous increase of spermatogonia to produce spermatocytes in drosophila hydei

The numbers of primary spermatocytes within cysts as well as numbers of postmeiotic spermatids in bundles in Drosophila hydei were determined. Within the contents of a single testis the cysts of primary spermatocytes are found to contain 5–11 germ cells. Furthermore, the number of spermatocytes per cyst is age-dependent, in that pupae have a mean of 8.1 cells whereas fertile adult males have a mean of 7.1 cells. Counts of spermatids in section of testes add further support to the view that the primary spermatocytes, from which the spermatids originated, were not formed in a strict geometric progression.     

Harmful B-chromosomes in a mealy bug: additional evidence

The B-chromosomes (B's; supernumeraries) of the mealy bug, Pseudococcus obscurus Essig, segregate preferentially into the two functional products of male meiosis. This segregation thus serves as an accumulation mechanism. A cytological study of a population from Oakland, California, confirmed the results obtained earlier that the B's are harmful and are maintained only because of their accumulation mechanism. The wild females were studied directly. The number of B's in the males was determined by analyzing ten or more daughters of females without B's (0B females) after these were inseminated by wild males. The 0B females were exposed to the wild males in screen cages. The analysis of 4732 daughters of 231 caged females indicated that among the males which inseminated these females, there were 19.9% 0B males and the mean number of B's was 1.46 ± 0.07. Among 224 wild females which were collected at about the same time there were 12.5% 0B females, and the mean number of B's was 1.88 ± 0.09. Since the frequencies of the B's in the population changed only slightly from generation to generation, the expected zygotes of this generation were assumed to be similar to those from which both the males and the females developed. The expected zygotes were calculated from the observed frequencies of the B's among the sperm and the known rates of transmission in females. The zygotes were very similar to the females but quite different from the males. It was concluded, therefore, that the B's had little or no effect on the females carrying them, but reduced the fitness of the males. The fitness of the 0B, 1B, 2B, 3B and 4B males was calculated to be 1.00, 0.64, 0.56, 0.38 and 0.20 respectively. The rate of transmission of the B's decreased with the increase in the number of B's, from 0.84 in 1B males to 0.51 in 4B males. This decrease, and the decrease in male fitness with the increase in the number of B's are expected to help stabilize the number of B's in the population.This paper is dedicated to Professor Sally Hughes-Schrader on the occasion of her seventy-fifth birthday.Supported by grants GB 1585 and GB 6745 from the National Science Foundation, Washington, D. C.     

Elimination of male germ cells in transgenic mice by the diphtheria toxin A chain gene directed by the histone H1t promoter

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.     

The Transcriptional Cell Atlas of Testis Development in Sheep at Pre-Sexual Maturity

Sheep testes undergo a dramatic rate of development with structural changes during pre-sexual maturity, including the proliferation and maturation of somatic niche cells and the initiation of spermatogenesis. To explore this complex process, 12,843 testicular cells from three males at pre-sexual maturity (three-month-old) were sequenced using the 10× Genomics Chromium^TM single-cell RNA-seq (scRNA-seq) technology. Nine testicular somatic cell types (Sertoli cells, myoid cells, monocytes, macrophages, Leydig cells, dendritic cells, endothelial cells, smooth muscle cells, and leukocytes) and an unknown cell cluster were observed. In particular, five male germ cell types (including two types of undifferentiated spermatogonia (A_pale and A_dark), primary spermatocytes, secondary spermatocytes, and sperm cells) were identified. Interestingly, A_pale and A_dark were found to be two distinct states of undifferentiated spermatogonia. Further analysis identified specific marker genes, including UCHL1, DDX4, SOHLH1, KITLG, and PCNA, in the germ cells at different states of differentiation. The study revealed significant changes in germline stem cells at pre-sexual maturation, paving the way to explore the candidate factors and pathways for the regulation of germ and somatic cells, and to provide us with opportunities for the establishment of livestock stem cell breeding programs.     

Germ line polysomy in the grasshopper Atractomorpha similis

Nineteen Eastern Australian populations of the grasshopper Atractomorpha similis (Acridoidea, Pyrgomorphidae) were sampled and male meiotic chromosomes, as well as some male and female somatic mitoses, were examined. In fourteen of these populations, a proportion of the males were found to carry between one and ten extra copies of a particular autosome, the megameric chromosome (A9). Numbers of extra chromosomes varied between but not within the individual follicles of the testis. The extra chromosomes were not found in somatic tissue. In all, 20% of males from the field were germ-line polysomic and within these males, 91% of germ cells were polysomic. In meiosis, extra copies of A9 present as univalents lagged at anaphase I or II and subsequently formed micronuclei which degenerated early in spermiogenesis. As one extra univalent is the most common polysomic condition in natural populations, this elimination of univalents suggests that most polysomic males produce a large proportion of normal haploid sperm. In laboratory cultures, selection for increased frequency of germ-line polysomy, conducted over four generations, raised the proportion of polysomic males from 23% to 71%. Selection against polysomy reduced its frequency to 5%. These breeding experiments also showed that germ-line polysomy is equally transmissible through both the male and the female parent. Transmission data also suggested that these extra chromosomes can arise de novo, presumably by unequal disjunction in previously diploid lines. A computer model was devised, simulating the effects of repeated non-disjunction over a series of mitotic divisions. The behaviour of this model suggested that the distributions of extra chromosome numbers observed in the laboratory generations most probably resulted from such a series of non-disjunctions, occurring in an initially diploid cell population. It seems, therefore, that the transmission of polysomy occurs through the agency of heritable factors which determine the probability of non-disjunction and thus the accumulation of a particular autosome during a specific series of mitotic divisions in the embryonic germ-line.     

Restriction of PGK-B to spermatogenic cells: Evidence from experimentally cryptorchid mice

The hypothesis that PGK-B, like LDH-C₄, is restricted to spermatogenic cells was explored by examining isozyme patterns in testes from mice depleted of germinal cells by surgical cryptorchism. In experimentally cryptorchized C57BL/10Sn males, decline in PGK-B activity paralleled decline in LDH-C₄ activity and was correlated with degeneration of spermatocytes, spermatids, and spermatozoa. Trace amounts of these sperm isozymes found in cryptorchid testes after the depletion of maturing germ cells probably came from degenerated spermatids and spermatocytes and not from somatic testicular cells.     

Modulation of MAA-induced apoptosis in male germ cells: role of Sertoli cell P/Q-type calcium channels

Spontaneous germ cell death by apoptosis occurs during normal spermatogenesis in mammals and is thought to play a role in the physiological mechanism limiting the clonal expansion of such cell population in the male gonad. In the prepubertal rat testis, the most conspicuous dying cells are pachytene spermatocytes, which are also the primary target of the apoptosis experimentally induced by the methoxyacetic acid (MAA). Since we have recently reported that Sertoli cells, the somatic component of the seminiferous epithelium, regulate not only germ cell viability and differentiation but also their death, we have further investigated the mechanism involved in such a control.     

Karyotype and chromosomal banding pattern in Heteropeza pygmaea

The chromosomes of somatic and germ line cells of female embryos produced by paedogenesis were studied. The haploid set in somatic cells consists of one long submetacentric chromosome, one large acrocentric, one medium metacentric and two small acrocentrics. The length vs arm index karyogram makes it possible to distinguish all but the two pairs of small acrocentric chromosomes. — Attempts were made to develope a method for banding pattern visualization. The best result was obtained using trypsin which induced banding in the chromosomes of the somatic cells and occasionally also of the germ line cells. The resulting banding patterns were frequently not identical in members of a chromosome pair. There was also a variation between metaphases within an embryo as well as from different embryos. Some tentative explanations for these results are discussed.     

Development of the larval ovary in the moth, Plodia interpunctella

The morphogenesis of ovaries and the organization of germ cells within them were visualized during the larval stages of the moth, Plodia interpunctella. The germ cells were observed by utilizing confocal microscopy coupled with immuno-fluorescent staining for the alpha-crystallin protein 25 (alphaCP25). The alphaCP25 was previously shown to be specific to germ cells of pupae and adults, and this study shows that alphaCP25 is present in larval germ cells as well. A cluster of 28 germ cells that stain for alphaCP25 was found in the gonads of newly hatched first instar larvae. The founding germ cells became segregated into four clusters, most likely by somatic cell intrusion, around the beginning of the second instar. Division of the primary germ cells began by the end of the second instar and the formation of all cystoblasts appeared to be completed within the four ovarioles by the end of the third instar. Within the ovarioles of third instar larvae, the germ cells were organized with a distal cap of seven germ cells which was segregated from the majority of the germ cells. The main body of germ cells was arranged around a central germ cell-free core as a spiral. Divisions of the cystoblasts to form cystocyte clusters were nearly completed during the fourth (last) larval instar. These features suggest that the strategy to produce follicles in moths is fundamentally different from the fruitfly, Drosophila. It appears that during the initial stages of ovary development in P. interpunctella, the primary germ cells undergo stage-complete divisions that are completed prior to the onset of the next set of divisions, which results in a complete complement of follicles available by the time of adult eclosion, while in Drosophila the primary germ cell divisions are initiated in the adult stage, and follicles are produced individually as resources are available.