Germ Cell Degeneration Research Articles

2,4,6-Trinitrotoluene-induced Reproductive Toxicity via Oxidative DNA Damage by its Metabolite

Several epidemiological studies and animal experiments showed that 2,4,6-trinitrotoluene (TNT), a commonly used explosive, induced reproductive toxicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of TNT on the male reproductive system in Fischer 344 rats. TNT administration induced germ cell degeneration, the disappearance of spermatozoa in seminiferous tubules, and a dramatic decrease in the sperm number in both the testis and epididymis. TNT increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in sperm whereas plasma testosterone levels did not decrease. These results suggest that TNT-induced toxicity is derived from direct damage to spermatozoa rather than testosterone-dependent mechanisms. To determine the mechanism of 8-oxodG formation in vivo, we examined DNA damage induced by TNT and its metabolic products in vitro. 4-Hydroxylamino-2,6-dinitrotoluene, a TNT metabolite, induced Cu(II)-mediated damage to 32 P-labeled DNA fragments and increased 8-oxodG formation in calf thymus DNA, although TNT itself did not. DNA damage was enhanced by NADH, suggesting that NADH-mediated redox reactions involving TNT metabolites enhanced toxicity. Catalase and bathocuproine inhibited DNA damage, indicating the involvement of H 2 O 2 and Cu(I). These findings suggest that TNT induces reproductive toxicity through oxidative DNA damage mediated by its metabolite. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by TNT and other nitroaromatic compounds.

Male germ cell death in mouse testes: possible involvement of Fas and Fas ligand.

Loss of germ cells is very common during various stages of mammalian spermatogenesis. Although cell death, particularly apoptosis, has been implicated, our understanding of the mechanisms underlying germ cell death is still limited. In order to elucidate the extent and mechanism of germ cell death, this review first covers what is known of germ cell degeneration in the normal testes of fetal, neonatal, and adult mice from electron microscopy (EM) and from terminal dUTP nick-end labeling (TUNEL) staining. The issue of whether the Fas and Fas ligand (FasL) system is involved in the induction of germ cell apoptosis in normal and damaged testes is then addressed, including consideration of both the ischemia-reperfusion model of testicular torsion and the estrogen-treated testis model of environmental endocrine disruption. Finally, this review proposes that different molecular pathways may be triggered to induce male germ cell apoptosis, depending upon the physiological and pathological states of the germ cells.

A repeated 28-day oral dose toxicity study of 17alpha-methyltestosterone in rats, based on the 'enhanced OECD Test Guideline 407' for screening the endocrine-disrupting chemicals.

As part of the international validation project to establish the Enhanced OECD Test Guideline 407, we performed a 28-day repeated-dose toxicity study of 17alpha-methyltestosterone, an exogenous androgen agonist. Special attention was paid to the sensitivity of additional parameters for detecting endocrine-related effects of endocrine-disrupting chemicals, based on the existing Test Guideline 407. Seven-week-old Crj:CD(SD)IGS rats were allocated to one of four groups, each consisting of ten males and ten females, and 17alpha-methyltestosterone was administered daily by gavage at doses of 0 (control), 5, 20 and 80 mg/kg body weight per day. Male rats were killed on the day after the 28th administration and females on the day of the diestrus stage during the 4 day period after the 28th administration. Male rats receiving 80 mg/kg 17alpha-methyltestosterone demonstrated decreases in testis and epididymis weights, atrophy of seminiferous tubules and Leydig cells, and degenerated pachytene spermatocytes in the testes and degenerated germ cells in the epididymides as major alterations. Female rats showed abnormal estrous cycles, decreases in ovary and adrenal weights, increase in immature follicles with decreased corpus lutea in the ovaries at doses of 5 mg/kg and higher, as well as atrophy of zona reticularis in the adrenals and increase in mammary gland secretion at 20 mg/kg and above. Dilatation of the lumina and apoptosis of endometrial cells in the uterus, mucinification in the vagina and increase in serum follicle-stimulating hormone were seen with 80 mg/kg. Among the parameters examined in the present experimental system, effects of 17alpha-methyltestosterone on endocrine-related organs were detected in organ weights and histopathological examination of both sexes, and in serum hormones and estrous cycle of females. Based on these results, the no-observed-adverse-effect level (NOAEL) in the present study was estimated to be below 5 mg/kg per day. In particular, effects were most sensitively detected by organ weights and histopathological examination of sexual organs.

Mitotic arrest of female germ cells during prenatal oogenesis. A colcemid-like, non-apoptotic cell death.

The sequence of events and a possible reason for germ cell death during oogenesis in the prenatal ovary were studied in rat and mouse embryos. ED 14-22 rat and ED 14-16 mouse embryos were studied using semithin sections for light microscopy and serial ultrathin sections for electron microscopy. In addition, the rat material was 3H-thymidine labelled for historadioautography and cytospin preparations of freshly obtained gonads were immunohistochemically analysed. During the transition from the proliferating oogonial stage to the meiotic prophase about 16% of the postmitotic oocytes do not pass the initial meiotic checkpoint on ED 18/19 in the rat (ED 15/16 in the mouse). These germ cells first show structural signs of mitosis; the diploid number of 'super-condensed' chromosomes are globally formed and are concentrated in the center of the cell. Although the germ cells show all morphological signs of living cells they never have mitotic spindles; the micro-tubulus-organisation-centres (MTOCs) are found peripherally and become concentrated, forming a single centrosomal body (acentriolar MTOC) as detected by immunohistochemistry for the centrosomal protein MPM2 and gamma-tubulin. EM studies show 25 nm tubule-like profiles within the MTOC bodies. The centrioles frequently lie separate from the MTOC material or are not present at all; the germ cells are apparently arrested in a prophase- or metaphase-like stage when they have reached the postmitotic G2/preleptotenal transition and are unable to enter meiosis. Forty-eight to 72 h after the first mitotically arrested germ cells are found, degeneration is seen in these germ cells. This second event, the germ cell death proper, shows neither criteria of apoptosis (cell shrinkage, marginal condensation of chromatin, DNA fragmentation) nor signs of necrosis (cell swelling, pycnosis, inflammation). Both arrested pro- and metaphase-like stages are found with signs of cell death and phagocytosis. The morphological signs of phagocytosis are found in neighbouring pregranulosa cells. The final heterocytotic bodies contain the remnants of the centrosomal (MTOC) material and DAPI-positive DNA material. The pregranulosa cells are mitotically silent during the phase when mitotic arrest and germ cell degeneration is found. The results suggest the presence of a hypothetical 'anti-spindle' factor, which under normal conditions is necessary for induction of meiotic prophase. The structural events of 'arrested mitosis' is reminiscent of those induced by the antimitotic, tubule-degrading drug colcemid. This type of arrest may inhibit meiosis of more than 33% prenatal germ cells and induce their cell death.

Spatial and temporal distribution of atrial natriuretic factor in the rat testis

The atrial natriuretic factor (ANF) is a cardiac hormone whose gene and receptor are widely expressed in extracardiac tissues and organs. ANF induces its biological effects by binding to its specific guanylyl-cyclase-A receptor, which synthesizes the intracellular second messenger cGMP. Increasing evidences indicate that the testis shows the highest reactivity of stimulation of guanylate cyclase by ANF. The well-established functionally active ANF receptors in seminiferous tubules raise the question of the origin and function of ANF in the testis. Therefore, the current study was carried out to investigate the spatial and temporal distribution of ANF in the rat testis by use of immunocytochemistry. Our immunocytochemical results showed that at different pre- and postnatal ages of testicular development ANF was constantly expressed in Leydig cell cytoplasm. However, the intensity of immunoreaction varied between the different Leydig cell populations (fetal, progenitor and immature) and apparently depends on the acquisition of testosterone producing ability. In seminiferous tubules ANF staining was established in the cytoplasm of the developing spermatocytes, in degenerating germ cells (23-day of age) in the cytoplasm of Sertoli cells, cap phase of acrosomal development and in the spermatids (55-day of age). The observed staining patterns suggest a broader spectrum of ANF activities and a possible participation in the whole process of regulation of germ cell development. Our data provide additional support for the hypothesis that ANF plays a major role in autocrine/paracrine regulation of the rat male gonad.

Ultrastructural analysis of chromatin defects in testicular spermatids in azoospermic men submitted to TESE-ICSI.

Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) is offered to treat obstructive and non-obstructive azoospermia, but factors that influence the outcome of ICSI are not well defined. The percentage of elongated spermatids with normal chromatin condensation in azoospermic patients submitted for TESE-ICSI was determined. The quantitative analysis could be applied to nine of 19 biopsies classified as incomplete late maturation arrest (LMA) and compared with 10 biopsies with normal spermatogenesis. The percentage of elongated spermatids with normal chromatin was lower in LMA than in normal histology (mean 4.4%, range 0-20, and mean 52.9%, range 40-70 respectively; P = 0.0001). The percentage of elongated spermatids with normal chromatin was negatively correlated with the serum concentration of FSH (r = -0.86, P < 0.0001) and the number of degenerated germ cells per 100 Sertoli cells nuclei (r = -0.68; P < 0.0001), while it was positively correlated with the number of elongating spermatids per 100 Sertoli cell nuclei (r = 0.81; P < 0.0001). The percentage of elongated spermatids with normal chromatin was not correlated with the rate of oocyte fertilization, while the delivery rate/cycle was higher in cases with normal histology compared with cases of LMA. These preliminary data suggest that an altered chromatin condensation is a ubiquitous defect in spermatids of non-obstructed azoospermic men submitted for TESE-ICSI.

Reduced fertility and spermatogenesis defects in mice lacking chromosomal protein Hmgb2.

High mobility group 2 protein (Hmgb2) is a member of the HMGB protein family, which includes the ubiquitous Hmgb1 and the embryo-specific Hmgb3. The three proteins are more than 80% identical at the amino acid level and their biochemical properties are indistinguishable. Hmgb1 is an abundant component of all mammalian nuclei and acts as an architectural factor that bends DNA and promotes protein assembly on specific DNA targets. Cells that lack Hmgb1 can survive, although mutant mice die shortly after birth. As Hmgb2 is present in all cultured cells and is abundant in thymus, the preferred source for HMGB proteins, it was considered a ubiquitous variant of Hmgb1. We show that in adult mice Hmgb2 is restricted mainly to lymphoid organs and testes, although it is widely expressed during embryogenesis. Mice that lack Hmgb2 are viable. However, male Hmgb2(-/-) mice have reduced fertility, that correlates with Sertoli and germ cell degeneration in seminiferous tubules and immotile spermatozoa. Significantly, Hmgb2 is expressed at very high levels in primary spermatocytes, while it is barely detectable in spermatogonia and elongated spermatids. This peculiar pattern of expression and the phenotype of mutants indicate that Hmgb2 has a specialised role in germ cell differentiation.

Inhibition of Fertility and Functional Alteration in the Genital Organs of Male Swiss Albino Mouse After Administration of Calotropis procera Flower Extract

Intramuscular administration of aqueous and ethanolic flower extracts of Calotropis procera (Ait.) R.Br. at doses of 10.0 or 5.0 mg/mouse/alternate day for a period of 20 days to sexually mature male albino mice induced functional sterility without loss of libido and sexual behaviour as evidenced by total inhibition of nidation in cycling parous female mice mated with the substance-treated males. The antifertility effect can be conjecturally attributed to the highly reduced sperm concentration and altered biochemistry of the genital organs. Glycogen, cholesterol and alkaline phosphatase content of the examined reproductive tissues exhibited a statistically significant increase in comparison to the control values, while sialic acid and acid phosphatase values were depleted. Also, the net weight of the testes and sex accessories were significantly reduced. Histologic profiles of the testes revealed extensive deleterious changes in the germinal tubules which contained mainly necrotic and degenerating germ cells. Further, the epididymal lumina appeared devoid of spermatozoa and exhibited mainly cell debris. The present results indicate that C. procera flower has a potent antispermatogenic activity in the albino mouse, but at the doses and experimental regimen employed, had no apparent effect on sexual behaviour or libido.

Expression of Hsp70-2 in unilateral cryptorchid testis of rhesus monkey during germ cell apoptosis.

We investigated the possible role of Hsp70-2 in germ cell apoptosis induced by heat stress in monkey unilateral cryptorchid testis. The study focused on in situ analysis of the testicular cell DNA fragmentation and on the possible relationship between Hsp70-2 expression and germ cell apoptosis. The TUNEL result showed that most of the germ cells were labeled in the cryptorchid testis on d 5 after induction of cryptorchidism; that with most of the apoptotic germ cells depleted, only a few germ cells were labeled on d 10; and that almost no apoptotic signal was observed in the cryptorchid testis on d 15 and thereafter. This indicates that the increasing germ cell degeneration in cryptorchid testis may take the form of apoptosis. Using in situ hybridization, immunohistochemistry, and Northern blot, we examined the changes of Hsp70-2 expression in the monkey cryptorchid testis. The level of Hsp70-2 mRNA decreased slightly, while the expression of HSP70-2 protein was almost unchanged at the early stage of germ cell apoptosis in the cryptorchid testis on d 5 and dropped dramatically along with the loss of apoptotic germ cells in the cryptorchid testis on d 10 after operation. It is therefore suggested that Hsp70-2 might not take part in inhibiting the apoptosis of germ cells at the early stage during operation-induced cryptorchid testis, and that Hsp70-2 gene does not belong to the immediate early related gene responsible for germ cell apoptosis induced by heat stress.

2-Bromopropane induced germ cell apoptosis during spermatogenesis in male rat.

2-Bromopropane (2-BP) causes testicular toxicity in humans and rats. However, the germ cell degeneration of testicular toxicity by 2-BP has not been understood. 2-BP at doses of 135, 405, and 1,355 mg/kg/day was daily injected subcutaneously into Sprague-Dawley rats for 28 days. At the dose of 1,355 mg/kg/day, 2-BP significantly decreased the weights of body and testes, eipididymis, seminal vesicle, and prostate, as well as daily sperm production. Atrophy of seminiferous tubules accompanied with degeneration of germ cells such as spermatogonia, spermatocytes, and elongated spermatids was observed in the testes of rats exposed to the 405 mg/kg/day and 1,355 mg/kg/day of 2-BP. TUNEL-positive germ cells were appeared in the 405 and 1,355 mg/kg/day of 2-BP-treated groups. In addition, ultrastructure alterations of apoptotic germ cells were observed by the electron microscopy study. Dead elongated spermatids were observed at 1,355 mg/kg/day after 28 days exposure. These results suggest that 2-BP impair spermatogenesis may result from apoptotic germ cell death.

Expression of endothelial nitric oxide synthase in the Sertoli cells of men with infertility of various causes.

To investigate how endothelial nitric oxide (eNOS) expression in the seminiferous tubules might be related to spermatogenesis, by examining eNOS expression in testicular tissue of patients infertile from various causes. The study included five fertile men with a normal sperm concentration, nine patients with obstructive azoospermia, 20 with varicocele testes and eight with idiopathic azoospermia (Sertoli cell-only syndrome). Testicular biopsy specimens were examined by immunohistochemistry for eNOS protein expression, in addition to a routine pathological assessment. eNOS protein was detected using an eNOS monoclonal antibody. A Sertoli cell staining index (SSI) was defined as the ratio of stained Sertoli cells per total number of Sertoli cells, and was compared among the groups. eNOS was localized to Sertoli cells in the seminiferous tubules and Leydig cells in the interstium; although some degenerating germ cells stained, normal germ cells did not. The SSI was significantly lower in patients with Sertoli cell-only syndrome than in either fertile men or patients with obstructive azoospermia or varicocele. However, the SSI did not correlate significantly with the Johnsen score. The expression of eNOS in Sertoli cells may depend on the existence of germ cells and be associated with germ cell development.

X-irradiation--induced changes in the progression of type B spermatogonia and preleptotene spermatocytes.

In response to induced DNA damage, proliferating cells arrest in their cell cycle or go into apoptosis. Ionizing radiation is known to induce degeneration of mammalian male germ cells. The effects on cell-cycle progression, however, have not been thoroughly studied due to lack of methods for identifying effects on a particular cell-cycle phase of a specific germ cell type. In this study, we have utilized the technique for isolation of defined segments of seminiferous tubules to examine the cell-cycle progression of irradiated rat mitotic (type B spermatogonia) and meiotic (preleptotene spermatocytes) G1/S cells. Cells irradiated as type B spermatogonia in mitotic S phase showed a small delay in progression through meiosis. Thus, it seems that transient arrest in the progression can occur in the otherwise strictly regulated progression of germ cells in the seminiferous epithelium. Contrary to the arrest observed in type B spermatogonia and in previous studies on somatic cells, X-irradiation did not result in a G1 delay in meiotic cells. This lack of arrest occurred despite the presence of unrepaired DNA damage that was measured when the cells had progressed through the two meiotic divisions.

Apoptotic germ-cell death and testicular damage in experimental diabetes: prevention by endothelin antagonism

This paper explores the role of endothelins (ETs) in diabetes-induced testicular damage by investigating, in a temporal manner, testes from streptozotocin (STZ)-induced diabetic rats. Testicular and epididymal weights and testicular morphology were assessed. Cell death was evaluated by light microscopy using conventional staining and morphology, and by apoptotic cell staining using the Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling (TUNEL) technique. Expression of endothelin-1 (ET-1) mRNA was evaluated by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Furthermore, effects of a mixed ETA and ETB receptor antagonist, bosentan, were studied. Testicular weights did not show any change at 1 month of follow-up, but were decreased after 6 months of diabetes. However, epididymal weights were significantly decreased at the end of both time periods in the diabetic rats. Morphological evaluations of the testes from diabetic rats showed a reduction in seminiferous tubular diameter, an increase in the number of empty testicular tubules and an increase in vascular density. Furthermore, degenerated germ cells and TUNEL-positive cells were significantly higher in diabetic rats than in control animals. The changes in diabetic animals were associated with increased ET-1 mRNA expression and were prevented by bosentan treatment. Administration of bosentan prevented decreased testicular weights, reduced seminiferous tubule diameters, increased vascular densities and incidences of degenerated and apoptotic germ cells and empty tubules in diabetic rats at the long-term follow-up. These results demonstrated that an ET-1 mediated pathway might be involved in testicular injury and germ-cell apoptosis in diabetes.

Apoptotic cell death and fertility in three unilateral cryptorchid rat models.

Three rat strains have been studied, using a sensitive apoptotic detection method for germ-cell degeneration, to resolve the controversy regarding the effect of cryptorchidism on the contralateral descended testis (CDT). Sprague Dawley and Buffalo rats were made cryptorchid by operation at 20-22 days of age, while trans-scrotal (T-S) rats were a congenitally unilateral cryptorchid strain. Sham operated rats or normal T-S littermates were used as controls. Experiments were performed over a period ranging from 2 weeks to 18 months. Testis weight was assayed, as was the detection of apoptosis by agarose gel laddering and immunohistochemistry by using the TUNEL method. Labeled cells in 150 cross-sectioned testis tubules were counted on the TUNEL stained slides and the mean number of labeled cells per tubule was calculated. Paternity studies on Sprague Dawley and T-S rats were carried out at 12 and 24 weeks of age to assess fertility by the resultant number of pregnancies and litter sizes. Both Sprague Dawley and T-S rat models showed a biphasic distribution of apoptosis levels. This biphasic distribution was not observed in Buffalo rats as they were only studied at later time points (12-20 weeks). A significant effect on either testis weight or apoptosis in the CDT compared with the control descended testis (P > or = 0.1) has not been found in these three cryptorchid models, and the present results are discussed with reference to observations of other researchers in rodents and humans. While the cryptorchid testis showed a high level of labeled apoptotic cells per tubule in all rat strains, fertility was not affected and remained the same as controls at 12 and 24 weeks. There was, however, a marked strain difference in fertility in T-S as compared with Sprague Dawley rats. After 24 weeks of cryptorchidism, both control and cryptorchid T-S rats had a 44% pregnancy incidence compared with a 90% pregnancy incidence in Sprague Dawley rats. In addition, litter size in T-S control and cryptorchid rats were small compared with those of Sprague Dawley rats at 12 and 24 weeks.

Disruption of spermatogenesis in boars sub-clinically infected with Trypanosoma brucei brucei

Data from 14 crossbred (Landreace × Large white) boars aged 10–12 months were used to investigate specific germ cells and to what extent Sertoli cells are prone to sub-clinical infection with strain Y58/98 Trypanosome brucei brucei and effects on spermatogenesis. Boars were divided into three groups, A, B and C of 5, 5 and 4 animals, respectively. Groups A and B were infected intraperitoneally with 2.8×10 6 trypanosomes per animal. Group C consisted of intact controls. At stable sub-clinical trypanosomiasis, boars in groups A and B together with two from the controls were weighed, scrotal circumferences were measured and animals were castrated on days 56 and 84 post infection, respectively. Testes were weighed. A portion of a testis was processed for histomorphometric assessment and another portion was used to determine gonadal sperm reserves by haemocytometry. Crude cells were converted to true cells. Sub-clinical trypanosomiasis was characterised by low live and testes weights, reduced scrotal circumference, scanty parasitemia peaks at long intervals and decreased libido. Histomorphometry of animals infected with T. brucei brucei revealed somniferous tubular distortion, denudation and or degeneration of germ cells and Sertoli cells leading to distortion of spermatogenesis. Spermatids and young primary spermatocytes were most prone to, while Sertoli cells and spermatogonia were least affected by sub-clinical trypanosomiasis. There was evidence of regeneration of germ cells from precursor stem cells, resulting in slightly increased gonodal sperm reserves as the post infection period increased. Infected boars may not attain original fertility levels consequently. It was concluded that boars in tropical regions that harbour endemic disease should be maintained under prophylactic conditions.

Deoxyribonucleic acid hypomethylation of male germ cells by mitotic and meiotic exposure to 5-azacytidine is associated with altered testicular histology.

Genomic methylation patterns originate during gametogenesis and are postulated to be involved in important developmental events, including gene regulation, embryogenesis, and genomic imprinting. In previous work, treatment of male rats with 5-azacytidine, a drug that blocks DNA methylation, resulted in abnormal embryo development when germ cells were exposed throughout spermatogenesis, encompassing mitotic, meiotic, and postmeiotic development, but not if they were only exposed postmeiotically. To explore the mechanisms underlying the effects of 5-azacytidine on sperm function, we determined the effects of the drug on testicular morphology, assessed whether exposure of meiotic spermatocytes resulted in abnormal pregnancy outcome, and examined the role of germ cell genomic demethylation in mediating the effects of 5-azacytidine on spermatogonia and spermatocytes. Male Sprague Dawley rats were treated three times a week with saline or 5-azacytidine (2.5 and 4.0 mg/kg) for 6 weeks (meiotic and postmeiotic germ cell exposure) and 11 weeks (mitotic, meiotic, and postmeiotic exposure). Six weeks of paternal treatment with the highest dose of 5-azacytidine resulted in an increase in preimplantation loss (corpora lutea minus implantation sites) without affecting testicular morphology or altering sperm DNA methylation levels. Eleven weeks of 5-azacytidine treatment at doses that cause preimplantation loss resulted in severe abnormalities of the seminiferous tubules, such as degeneration and loss of germ cells, atrophy of seminiferous tubules, presence of multinuclear giant cells, and sloughing of immature germ cells into the lumen, and a 22-29% decrease in genomic methylation levels in epididymal sperm. On closer evaluation of testicular histology using terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling detection in situ, both 6 and 11 weeks of 5-azacytidine treatment resulted in an increase over the control value in the number of apoptotic germ cells in the seminiferous tubules. Analysis of DNA methylation levels in isolated germ cells of treated males indicated that spermatogonia were more susceptible to the hypomethylating effects of 5-azacytidine than were spermatocytes. These studies provide evidence of an association between demethylation of germ cell DNA and alterations in testicular histology.

Meiosis and apoptosis in germ cells of X‐autosome translocation carrier boars

Meiotic features and fate of germ cells were studied using electron microscopy on surface spread spermatocytes and in situ tests for apoptosis on testicular tissues of normal boars and X-autosome translocation (X-AT) carrier boars. Histological sections of the translocation t(Xp+; 14q−) carrier boars showed accumulation of degenerating germ cells including binucleate and multinucleate cells, as well as pyknosis and nuclear fragmentation characteristic of apoptosis. Synaptonemal complex analysis of X-AT carrier boars revealed 19 bivalents including a large complex made up of the altered X (Xp+) and normal chromosome 14, and a smaller element representing the Y chromosome in synapsis with the derived chromosome 14 (14q−) in most (89.3%) of the germ cells. In situ tests for apoptotic DNA fragmentation revealed positive signals exclusively among early spermatocytes and degenerating germ cells. These findings and the absence of stages beyond pachytene suggest that the meiocytes are arrested at pachytene and eliminated through apoptotic process in spite of the complete synapsis displayed by the chromosomes involved in this translocation. Failure of meiotic progress in our X-AT carriers would appear to be the result of the disruption of gene sequence (or function) caused by the involvement of the X chromosome in this rearrangement, rather than the deleterious consequences of abnormal segregation anticipated in reciprocal translocation carriers. We hypothesize that this disruption could have affected the induction of stage-specific gene products in meiosis such as heat shock proteins and caused the excessive release of endonucleases normally produced by early prophase meiocytes, leading to their apoptosis in our X-autosome translocation carrier boars. Mol. Reprod. Dev. 56:448–457, 2000. © 2000 Wiley-Liss, Inc.

Efficiency of spermatogenesis: a comparative approach

Efficiency of spermatogenesis is the estimated number of spermatozoa produced per day per gram of testicular parenchyma. Spermatogenesis is the process of cell division and cell differentiation by which spermatozoa are produced in testes. Efficiency of spermatogenesis is influenced by species differences in the numerical density of germ cell nuclei and in the life span of these cells. Activities of spermatogonia, spermatocytes, and spermatids partition spermatogenesis into three major divisions (spermatocytogenesis, meiosis, and spermiogenesis, respectively). Spermatocytogenesis involves mitotic germ cell division to produce stem cells and primary spermatocytes. Meiosis involves duplication of chromosomes, exchange of genetic material, and two cell divisions that reduce the chromosome number and yield four spermatids. In spermiogenesis, spherical spermatids differentiate into mature spermatids which are released in the lumen of seminiferous tubules as spermatozoa. Spermatogenesis and germ cell degeneration can be quantified from numbers of germ cells in various developmental steps throughout spermatogenesis. Germ cell degeneration occurs throughout spermatogenesis; however, the greatest impact occurs during spermatocytogenesis and meiosis. There are species and seasonal influences on the developmental steps in spermatogenesis at which germ cell degeneration occurs. Number of Sertoli cells, amount of smooth endoplasmic reticulum of Leydig cells, and the number of missing generations of germ cells within the spermatogenic stage of the cycle influence efficiency of spermatogenesis. Efficiency of spermatogenesis is influenced to the amount of germ cell degeneration, pubertal development, season of the year, and aging of humans and animals.

Induction and inhibition of testicular germ cell apoptosis by fluoroacetate in rats.

Fluoroacetate (FA), an inhibitor of aconitase, is known to lower the intracellular level of adenosine triphosphate (ATP), which recently has been suggested to be a possible determinant of the form of cell death, apoptosis or necrosis. To investigate which form of germ cell death occurs in FA-induced testicular toxicity, adult Sprague Dawley rats were given a single oral dose of FA (0.5 or 1.0 mg/kg) and euthanized at 3, 6, 12, 24, 48, and 72 h thereafter. Germ cell degeneration was histologically first found in early round spermatids at stage I and in spermatogonia at stages II-IV of seminiferous tubules 6 and 12 h, respectively, after dosing. Degenerating spermatogonia exhibited characteristic features of apoptosis as demonstrated by both electron microscopy and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), whereas spermatids did not. At the 24 and 48 h time points, degenerating spermatids were continually present and subsequently formed multinucleated giant cells, while the number of degenerating spermatogonia and TUNEL-labeled spermatogonia was drastically and/or significantly decreased compared to those from the control group, indicating that spontaneous male germ cell apoptosis is inhibited. Coincident with these morphological changes, DNA laddering on gel electrophoresis was apparent only 12 h after dosing. The results demonstrate that FA induces either apoptosis or necrosis of male germ cells in the early stage after dosing and subsequently inhibits spontaneous apoptosis.

Meiosis and apoptosis in germ cells of X-autosome translocation carrier boars.

Meiotic features and fate of germ cells were studied using electron microscopy on surface spread spermatocytes and in situ tests for apoptosis on testicular tissues of normal boars and X-autosome translocation (X-AT) carrier boars. Histological sections of the translocation t(Xp+; 14q-) carrier boars showed accumulation of degenerating germ cells including binucleate and multinucleate cells, as well as pyknosis and nuclear fragmentation characteristic of apoptosis. Synaptonemal complex analysis of X-AT carrier boars revealed 19 bivalents including a large complex made up of the altered X (Xp+) and normal chromosome 14, and a smaller element representing the Y chromosome in synapsis with the derived chromosome 14 (14q-) in most (89.3%) of the germ cells. In situ tests for apoptotic DNA fragmentation revealed positive signals exclusively among early spermatocytes and degenerating germ cells. These findings and the absence of stages beyond pachytene suggest that the meiocytes are arrested at pachytene and eliminated through apoptotic process in spite of the complete synapsis displayed by the chromosomes involved in this translocation. Failure of meiotic progress in our X-AT carriers would appear to be the result of the disruption of gene sequence (or function) caused by the involvement of the X chromosome in this rearrangement, rather than the deleterious consequences of abnormal segregation anticipated in reciprocal translocation carriers. We hypothesize that this disruption could have affected the induction of stage-specific gene products in meiosis such as heat shock proteins and caused the excessive release of endonucleases normally produced by early prophase meiocytes, leading to their apoptosis in our X-autosome translocation carrier boars.