Primary Spermatocyte Stage Research Articles

Positional cloning of the Hybrid sterility 1 gene: fine genetic mapping and evaluation of two candidate genes

The Hybrid sterility 1 (Hst1) gene affects fertility of male hybrids between certain laboratory strains (such as C57BL/10) and some Mus musculus musculus mice by causing a breakdown of spermatogenesis at the stage of primary spermatocytes. In the process of positional cloning of the Hst1 gene, we generated a contig of bacterial artificial chromosomes and subsequently a low coverage sequence of the candidate region of the 129S1/SvImJ strain. Development of new genetic markers allowed us to narrow the Hst1 region from 580 to 360 kb. The products of two genes from this region, TATA-binding protein (Tbp) and proteasome subunit beta 1 (Psmb1), accumulate during spermatogenesis. These proteins have been described previously as having conserved C-terminal sequences and species-specific N-termini. We evaluated the candidacy of these genes for Hst1 by allelic sequencing and by real-time semiquantitative reverse-transcription PCR of testicular mRNAs.

Influence of stress on gonadotrophin induced testicular recrudescence in the lizardMabuya Carinata

Administration (ip) of FSH (10 IU/0.1 ml distilled water (dw)/lizard/alternate days/30 days) to adult male lizards, Mabuya carinata, during the early recrudescence phase of the reproductive cycle caused activation of spermatogenic and steroidogenic activity of the testis, as shown by a significant increase in mean number of spermatogonia, primary spermatocytes and spermatids, and serum levels of testosterone, as compared to initial controls. In addition, there were abundant spermatozoa in the lumen of the seminiferous tubules. Interestingly, administration of a similar dosage of FSH to lizards exposed to stressors (handling, chasing, and noise randomly applied, five times a day for 30 days) resulted in a significant increase in mean number of spermatogonia and primary spermatocytes over initial control values, whereas the number of secondary spermatocytes and spermatids and serum levels of testosterone did not significantly differ from those of initial controls, and were significantly lower than FSH treated normal lizards. Further, spermatozoa were infrequently found in the seminiferous tubules of these lizards. Treatment controls (receiving 0.1 ml dw/lizard/alternate days for 30 days) did not show significant variation in mean number of spermatogonia, spermatocytes and spermatids, and serum levels of testosterone from initial controls. Another group of lizards was exposed to stressors and did not receive FSH. These lizards showed a significant decrease in mean number of secondary spermatocytes compared to treatment controls and all other parameters did not significantly differ from those of both control groups. The results reveal that gonadotrophin-induced spermatogonial proliferation occurs under stressful conditions, whereas progress of spermatogenesis beyond primary spermatocyte stage is impaired due to inhibition (under stress) of gonadotrophin induced steroidogenic activity in M. carinata.

Drosophila TGIF is essential for developmentally regulated transcription in spermatogenesis.

We have investigated the role of TGIF, a TALE-class homeodomain transcription factor, in Drosophila development. In vertebrates, TGIF has been implicated, by in vitro analysis, in several pathways, most notably as a repressor modulating the response to TGFbeta signalling. Human TGIF has been associated with the developmental disorder holoprosencephaly. Drosophila TGIF is represented by the products of two tandemly repeated highly similar genes, achintya and vismay. We have generated mutations that delete both genes. Homozygous mutant flies are viable and appear morphologically normal, but the males are completely sterile. The defect lies at the primary spermatocyte stage and differentiation is blocked prior to the onset of the meiotic divisions. We show that mutants lacking TGIF function fail to activate transcription of many genes required for sperm manufacture and of some genes required for entry into the meiotic divisions. This groups TGIF together with two other genes producing similar phenotypes, always early and cookie monster, as components of the machinery required for the activation of the spermatogenic programme of transcription. TGIF is the first sequence-specific transcription factor identified in this pathway. By immunolabelling in mouse testes we show that TGIF is expressed in the early stages of spermatogenesis consistent with a conserved role in the activation of the spermatogenesis transcription programme.

Using the male gamete for assisted reproduction: past, present, and future.

Using the male gamete for assisted reproduction: past, present, and future.

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.

Human recombinant stem cell factor promotes spermatogonial proliferation, but not meiosis initiation in organ culture of newt testis fragments

We previously showed that mammalian FSH stimulates the proliferation of newt spermatogonia and induces their differentiation into primary spermatocytes in vitro. In the current study, to examine a possibility that stem cell factor (SCF) is involved in the proliferation of newt spermatogonia and/or their differentiation into primary spermatocytes, human recombinant SCF (rhSCF) was added to organ culture of testicular fragments. rhSCF was found to stimulate the spermatogonial proliferation and the spermatogonia progressed to the seventh generation that is the penultimate stage before primary spermatocyte stage. However, the spermatogonia did not differentiate into primary spermatocytes, but instead died of apoptosis. These results indicate that rhSCF promotes the proliferation of newt spermatogonia, but not the initiation of meiosis.

Reactivity of Germ Cell Maturation Stage-Specific Markers in Spermatocytic Seminoma: Diagnostic and Etiological Implications

It is generally accepted that testicular seminomas and spermatocytic seminomas have separate pathogeneses, although the origin of these two types of germ cell tumors of the adult testis remains a matter of debate. Although an embryonic germ cell origin seems to be most likely for seminomas, a spermatogonia-spermatocyte origin has been suggested for spermatocytic seminoma. To shed more light on the etiology of spermatocytic seminomas, we undertook an immunohistochemical and molecular approach using SCP1 (synaptonemal complex protein 1), SSX (synovial sarcoma on X chromosome), and XPA (xeroderma pigmentosum type A) as targets. Although a stage-specific expression pattern has been reported for SCP1 and SSX in normal spermatogenesis, we demonstrate here that it also exists for XPA. In fact, immunohistochemistry shows that the proteins of SCP1 and XPA are specifically present in the stage of primary and pachytene spermatocytes. In contrast, SSX was found in spermatogonia and primary spermatocytes, as well as in germ cells, from at least the 17th week of intrauterine development onward. Although no protein encoded by any of these genes was detected in tumor cells of a series of testicular seminomas, all tested spermatocytic seminomas were positive, in agreement with expression analysis. These data support the model that seminomas originate from an embryonic germ cell, and they imply that the cell of origin of spermatocytic seminomas is at least capable of maturing to the stage of spermatogonia-pachytene spermatocyte.

Cytogenetical and histological studies in testis of Tayassu tajacu (Cateto), Tayassu pecari (Queixada) and a natural interspecific hybrid

The karyotype, the histological structure of the testis and the synaptonemal complex (SC) of the mammalian species Tayassu tajacu, Tayassu pecari and of an interspecific male hybrid captured in nature were analysed. The specimens of T. tajacu (2n=30) and T. pecari (2n=26) exhibited seminiferous tubules with germ cells in all spermatogenesis stages. In the SC studies both species had a regular structure, easily identified in the autosomes and in the sex chromosomes. The hybrid (2n=28) had seminiferous tubules with almost all germinal cells in the spermatogonium stage and only a few cells in the primary spermatocyte stage. Gross abnormalities in SC were observed. A few lateral elements showed regular or partially regular synapsis, other lateral elements were synapsed as multivalents, and most axial elements remained unsynapsed. The results suggest that the karyotypes of the parental species have sufficient differentiation to avoid chromosome synapsis. Alternatively, the hypothesis of the existence of genetic incompatibilities between the parental species is discussed.

Testicular development, histology, and hormone profiles in three yearling angus bulls with spermatogenic arrest

This article discusses the interactions between testis criteria and hormone profiles in Angus bulls with spermatogenic arrest. From 2 to 12 months (mo), testis diameter and hormone concentrations (basal and GnRH-stimulated) were evaluated in 27 bulls. At 12 mo, testes were excised. The z statistical test was used to determine whether parameters in three infertile bulls were different (P < 0.05) from those in 24 bulls with normal spermatogenesis. Bull 1 had Sertoli cell-only syndrome and Bull 2 had 90% of the tubules without germ cells and only A1 spermatogonia in the remaining. In Bull 3, germ cells did not advance beyond the primary spermatocyte stage. At 12 mo, testes of Bull 1 (99 g), Bull 2 (105 g) and Bull 3 (32 g) weighed less than those of normal bulls (251.5 ± 56 g). Sertoli cell numbers/testis in Bull 1 (3.8 × 10 9) and Bull 2 (4.3 × 10 9) were not different from those in normal bulls (4.9 ± 0.3 × 10 9), but were reduced in Bull 3 (1.6 × 10 9). The number of Leydig cells per gram of testis parenchyma was higher in Bull 1 (5.4 × 10 7), Bull 2 (7.3 × 10 7) and Bull 3 (19 × 10 7) than in normal bulls (3.6 ± 0.2 × 10 7). In Bulls 1 and 2, basal and GnRH-stimulated LH, FSH, testosterone (T), androstenedione (Δ 4A) and estradiol 17-β (E 2) were within normal ranges at most ages. However, basal FSH and LH were greater in Bull 3 than in normal bulls, probably the causes for higher Leydig cell density. Also in the same animal, GnRH induced lower responses in LH and FSH, consequence of low basal T and E 2 at some ages. Basal and GnRH-stimulated Δ 4A in Bull 3 were greater than in normal bulls after 6 mo, indicating impairment of Leydig cell differentiation. Deficiency in hormone secretion did not appear to be the cause of infertility, which points toward impaired gonadal responses or secretion of intratesticular factors, or genetic defects. Moreover, infertile animals may not always show pronounced changes in hormone secretion, but evaluation of testis growth around puberty can help identify those animals that do not proper gonadal development.

Immunohistochemical analysis of cyclins in canine normal testes and testicular tumors.

The expression of cyclins A, D1, D2 and E were examined immunohistochemically in 5 canine normal testes and 31 testicular tumors, including 14 seminomas, 11 Sertoli cell tumors and 6 Leydig cell tumors. In canine normal testes, cyclin A expression was detected in spermatogonia and primary spermatocytes. This suggests that A-type cyclins may play some role in canine spermatogenesis. Cyclin A expression was also observed in 13/14 (92.9%) seminomas and 2/11 (18.2%) Sertoli cell tumors, but no positive reaction was observed in Leydig cell tumors. Parallel examinations for cyclins D1, D2 and E gave negative results in canine normal testes and testicular tumors. High levels of cyclin A expression in canine seminomas indicate that the neoplastic germ cells may be arrested at the spermatogonia and primary spermatocyte stages of differentiation.

Immature germ cell conception-in vitro germ cell manipulation.

Experimental studies in laboratory animals have shown that successful conception can be achieved by fertilizing oocytes with immature male germ cells. This gave rise to the concept that immature germ cells recovered from the testes of azoospermic men with maturation arrest may be used for assisted reproduction. However, in contrast to using germ cells recovered from healthy animals, clinical application to the treatment of male sterility is burdened by inherent defects in germ cells attributable to underlying testicular pathology. The recent introduction of in vitro germ cell culture/manipulation techniques makes it possible, in some cases, to overcome the in vivo maturation arrest by allowing an additional meiotic and post-meiotic differentiation and the selective harvesting of cells devoid of apoptosis-related nuclear and cytoplasmic damage. These techniques enabled the first births of normal infants fathered by azoospermic men with maturation arrest at the primary spermatocyte stage and improved the efficacy of assisted reproduction in men with maturation arrest at the round spermatid stage.

Effects of Hormones on Spermatogenesis in Men with Histological Diagnosis of Spermatogenic Arrest at the Primary Spermatocyte (PS) Stage or Sertoli Cell-Only Syndrome (SCOS)

Objectives: Men with absence of spermatozoa in the therapeutic testicular biopsy (TTB) material represent the most difficult cases of non-obstructive azoospermia (NOA). Even when alive round spermatids (RSs) are present in the TTB samples of the latter men, the outcome of ooplasmic injections of RSs rarely alleviates the couple’s infertility. We evaluated the effects of a combined administration of Profasi (Serono Co.) and Gonal-F (Serono Co.) on testicular spermatogenic and steroidogenic function. Design: Diagnostic testicular biopsy (DTB; hematoxylin-eosin stain) outcome and TTB (mincing testicular tissue and processing it for assisted reproduction trial) outcome were compared before and after hormonal treatment in three groups of NOA-men. Materials and Methods: All men had peripheral serum LH and FSH values less than twice the upper normal level of each respective hormone. TTB was performed at least four months after the performance of DTB. At the time of DTB, intratesticular testosterone (ITT) was measured, whereas, at the time of TTB, fractions of dispersed cells were processed for fluorescent in situ hybridization (FISH) techniques or observation via transmission electron microscope (TEM). Gonal-F (50 IU three times a week) and Profasi (2,500 IU twice a week) were administered for 12–24 months in a) 21 men whose DTB showed arrest at the PS stage and the TTB revealed absence of RSs and spermatozoa (group A), b) 7 men whose DTB demonstrated arrest at the PS stage and the TTB was positive for RSs but negative for spermatoa/elongating/elongated spermatids (group B), and c) 5 men whose DTB showed SCOS and the TTB demonstrated few PSs but not RSs (group C). New DTB, TTB, FISH, and TEM techniques were performed at the end of the treatment. Results: At the end of the homonal treatement, DTB images showed the same level of spermatogenic arrest as prior to treatment in all men of groups A, B, and C. Peripheral serum testosterone increased in all the participants. Observations of TTB samples post-treatment demonstrated few RSs in 8 men of group A. One of the latter 8 men showed spermatozoa, as well. No RSs were observed in TTB-samples of group C post-treatment. TEM showed elongating or elongated spermatids having tail length 0.004–0.047 mm in the TTB materials of 5 men of group B post-treatment. However, testicular spermatozoa were found in only two of the latter men. When the groups A and B were analyzed together it was found that the 13 men who responded to the treatment (development of RSs in the second TTB for group A; spermatid elongation in the second TTB for group B) had (prior to the hormonal treatment) significantly lower (P<0.05; Wilcoxon’s test) ITT profiles than the 15 remaining men who did not respond to the treatment. There was a large variation in ITT profiles prior to the treatment in groups A and B (taken together the groups A and B: 76–2675 ng/g testis). Four out the five men of group B who responded to the treatment had relatively low ITT profiles (less than 300 ng/g testis) pre-treatment. Conclusions: Hormonal treatment in men with early maturation arrest stimulates spermatogenesis in a clinically significant percentage (46%). Prior to the hormonal administration, the presence of RSs in the TTB of men with histological diagnosis of early maturation arrest or small ITT levels indicates good prognosis for response to the treatment. Hormonal treatment in SCOS-men without foci of RSs/spermatozoa is not recommended. This research was supported by a grant from the JG Fertility Institute.

In-vitro maturation of immature human male germ cells

Both meiotic and postmeiotic maturation events have been observed to occur in human male germ cells during in-vitro culture. The temperature of 30°C, medium supplementation with follicle-stimulating hormone and testosterone and the maintenance of the original cell–cell associations within explanted segments of the testicular seminiferous tubules are common features of the most efficient culture systems. The in-vitro maturation processes are markedly accelerated as compared to the in-vivo situation, probably due to the abrogation of a checkpoint controlling the full assembly of molecules needed for spermiogenesis. Moreover, both meiotic and postmeiotic maturation processes can be reactivated in vitro in some cases with a complete block of the same processes in vivo. Healthy babies were born after micromanipulation-assisted fertilization with in-vitro matured elongated spermatids from men with complete in-vivo maturation arrest at the round spermatid stage and at the primary spermatocyte stage.

Prognostic factors for successful testicle spermatid recover

Men with non-obstructive azoospermia exhibit different histopathologic syndromes in the testicle biopsy, varying from aplasia, Sertoli cell only syndrome, maturation arrest and hypoplasia. The genetic basis of these syndromes is discussed. We present the diagnostic testicle biopsies performed on 160 consecutive non-obstructive azoospermic patients, and these results were correlated with the findings after multiple bilateral treatment testicle biopsy. Each syndrome had to be reevaluated as for the presence of at least one focus of spermatogenesis up to the primary spermatocyte, round spermatid, elongating spermatid, elongated spermatid, or spermatozoa stages. The clinical outcome using donor sperm-IVF, spermatid or sperm intracytoplasmic injection is thereafter presented. A new prognosis based on the findings of this large clinical series coupled to results obtained with Y chromosome molecular screening is offered. Alternative treatments to donor sperm for men without spermatids are discussed.

In-vitro spermatogenesis resumption in men with maturation arrest: relationship with in-vivo blocking stage and serum FSH.

We have shown previously that germ cells recovered from some men with maturation arrest can resume spermatogenesis in vitro and give rise to late elongated spermatids. This study relates the ability of germ cells to differentiate in vitro to the stage at which spermatogenesis is blocked in vivo and to the patient's serum FSH concentration. The presence of germ cells at different stages of spermatogenesis was assessed, before and after culture, by classical cytology, by fluorescence in-situ hybridization and by immunocytochemistry with a germline-specific marker. The proportion of cases of maturation arrest at the primary spermatocyte, secondary spermatocyte and spermatid stage in which in-vitro resumption of meiosis was achieved was 24.3% (9/37), 100% (3/3) and 51.1% (23/45) respectively. Serum FSH concentrations were higher than normal in most cases. However, lower values were measured in patients in whom in-vitro spermatogenesis was achieved compared with those in whom no progression was detected. These data show that, under the conditions of this study, germ cells from men with very high serum FSH concentrations (>20 IU/l) are less likely to resume spermatogenesis in vitro than those coming from men with only moderate increase (10-20 IU/l).

Efficiency of the process of meiosis in scrotal testes of healthy boars and unilateral abdominal cryptorchid boars.

Unilateral abdominal cryptorchidism has usually been correlated with abnormalities in the spermatogenic activity of the scrotal testis. The present study describes the effects of unilateral abdominal cryptorchidism on the meiotic process in scrotal testes from postpubertal boars. The percentage of primary spermatocytes, secondary spermatocytes, and round spermatids was evaluated in testicular smears from scrotal testes of healthy boars and of right-sided unilateral abdominal cryptorchid boars. As compared to the scrotal testes of healthy boars, the scrotal testes of unilateral abdominal cryptorchid boars showed low transformation from primary to secondary spermatocytes (meiosis I), but normal transformation from secondary spermatocytes to round spermatids (meiosis II). The data obtained indicate that spontaneous unilateral abdominal cryptorchidism on the right side induced partial arrest of spermatogenesis at the primary spermatocyte stage that was attributed to anomalies in Sertoli-cell activity. Abnormal paracrine signals from altered Sertoli cells could have resulted in either disturbed mitosis, which led to the formation of spermatocytes with an abnormal DNA content, or abnormalities in the metabolic activity and the organization of the cytoskeleton of primary spermatocytes.

O-062. In-vitro maturation of human male germ cells: a new treatment for maturation arrest at the primary spermatocyte stage

O-062. In-vitro maturation of human male germ cells: a new treatment for maturation arrest at the primary spermatocyte stage

RADHA--a new male germ line-specific chromosomal protein of Drosophila.

A new chromosomal protein - RADHA - of Drosophila is described that is specific for the male germ line. It is encoded by a single-copy gene, located in the region 96C-D of D. melanogaster polytene chromosomes. Transcription of the radha gene is restricted to the primary spermatocyte stage. The protein initially accumulates in some of the Y-chromosomal lampbrush loops. After meiosis it is found in the nuclei of spermatids and might be involved in chromatin rearrangement processes in the male germ line. RADHA is a basic protein with a C-terminal leucine zipper region and several segments capable of forming coiled-coil structures.

HIV in testis: quantitative histology and HIV localization in germ cells

The testes of AIDS patients invariably show decreased spermatogenesis and are atrophic. These testicular changes can be grouped into three categories: (1) spermatogenesis present, but decreased; (2) spermatogenic arrest at primary spermatocyte stage; and (3) Sertoli only (or almost Sertoli only). The purpose of this study is 2-fold: firstly, to quantitate the numbers and types of germ cells in these three groups as compared with normals. In addition the presence of HIV-1 DNA positive germ cells was quantitated by PCR in situ hybridization. HIV-1 was identified in 14 of 15 testes from HIV infected adults, and was present in 25–33% of residual germ cells. There was an average of 18.9 HIV infected germs cells/tubule in the spermatogenesis group, 6.3 HIV infected germs cells in the spermatogenic arrest group, and 0.25 HIV infected germ cells in the almost Sertoli only group. HIV-1 DNA was absent in three of the three preadolescent boys testes (HIV acquired in utero). This study quantitates the degree of germ cell loss in AIDS patients and quantitates the degree of HIV positivity of the residual germ cells, thus shedding more light on the testicular HIV burden, with its possible repercussions for sexual transmission of HIV.