Testicular development in Brahman bulls

Brahman breed bulls (Bos indicus) are widely used to introduce environmental resistance traits into meat-producing herds. However, their reproductive development is slower than European breeds (Bos taurus). The objective of this study was to assess the development of the seminiferous epithelium in Brahman bulls. Twenty-three prepubertal bulls were castrated and testicular samples taken for histological processing. Light microscopic images were digitized and cells of the seminiferous epithelium were assessed. Immature Sertoli cells gradually decreased in numbers and were no longer detected after approximately 14 months of age; concurrently, the numbers of mature Sertoli cells increased from 10 to 14 months. Spermatogenesis started during the ninth month; prior to that, only gonocytes and immature Sertoli cells were observed. Type A spermatogonia, spermatocytes, round spermatids, elongated spermatids and spermatozoa were first detected at 9.5, 11, 11, 13 and 16 months of age, respectively. The delay in the onset of puberty in Brahman bulls with respect to B. taurus was attributed to a longer duration of the prepubertal period (interval from start of spermatogenesis to puberty) and a later start of spermatogenesis.     

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

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

The development of puberty and sexual maturity in the Australian Swamp buffalo bull

Testes from 47 juvenile Swamp buffalo bulls were examined for puberty and sexual maturity histologically and daily sperm production per gram of testis parenchyma was determined by enumeration of elongated spermatids in homogenates of testis parenchyma. Puberty was defined as the attainment of a daily sperm production per gram of testis parenchyma >0.5 x 10(6). In most bulls, puberty is attained by 24 mo of age, when scrotal circumference (SC) is approximately 16 cm, and liveweight exceeds 135 kg. Sexual maturity was defined as the attainment of adult levels of daily sperm production per gram of testis parenchyma (14 x 10(6)). In most bulls, this occurs at 30 to 33 mo of age, when SC is in the 17-to 20-cm range, and liveweight generally exceeds 250 kg. There was marked individual variation in age, liveweight and SC at both puberty and sexual maturity.     

Developmental changes of DNA ligase during ram spermatogenesis

The molecular forms and activities of ram DNA ligase have been investigated during spermatogenesis from the stage of early round spermatids to ejaculated spermatozoa. Through germ cell maturation, two consecutive forms of the enzyme (6S and 7S) have been found. The 6S form (DNA ligase II) is observed in primary and secondary spermatocyte, as well as in round spermatids. The 7S form (DNA ligase I) is present in elongated spermatids and in the sole round cell population with spermatogonia and young primary spermatocytes. In ram germ cells, DNA ligase I and DNA ligase II appear to be respectively associated with DNA replication repair. The absence of DNA ligase II associated with the absence of DNA repair in testicular and ejaculated spermatozoa might be related to male infertility.     

Rat spermatogenic cell beta-D-galactosidase: characterization, biosynthesis, and immunolocalization

In this study we have demonstrated that the rat sperm acrosomal beta-d-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain beta-galactosidase and four other glycohydrolases (beta-d-glucuronidase, alpha-d-mannosidase, alpha-l-fucosidase, and beta-N-acetylglucosaminidase). With the exception of alpha-l-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of beta-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t(1/2) of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive.     

Preparation of spermatogenic cell populations at specific stages of differentiation in the human.

Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin-DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enriched in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2-4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.     

Inhalation exposure-rate of ethylene oxide affects the level of DNA breakage and unscheduled DNA synthesis in spermiogenic stages of the mouse

The effect of ethylene oxide (EtO) inhalation-exposure rate on the induction of DNA breakage in late spermatids and on unscheduled DNA synthesis (UDS) in early spermatids was studied. The exposures were 450 parts per million (ppm) for 4 h, 900 ppm for 2 h, and 1800 ppm for 1 h. Thus, the total exposure was always 1800 ppm-h. Both DNA breakage and UDS were found to increase by a factor of approximately 3 in going from the low to high EtO concentration, suggesting that the molecular dose of EtO to the testis had increased by a similar factor. Our results are consistent with the EtO exposure-rate effect found by Generoso et al. (1986) for induction of dominant-lethal mutations in late spermatids and early spermatozoa.     

Bilateral asymmetry in skeletal growth and maturation as an indicator of environmental stress.

This study examined the efficacy of bilateral asymmetry in epiphyseal union as an indicator of environmental stress affecting the skeleton. We compared the extent of asymmetry in the postcranial skeleton between two cemetery samples excavated from Medieval Kulubnarti, Sudanese Nubia. Past studies have strongly suggested that these ancient Nubians experienced environmental stress-the early Christian period (550-750 AD) population to a greater extent than the late Christian period (750-1450 AD) population. We hypothesized that if bilateral asymmetry is a reflection of stress, then it should be present or greater in the more stressed population, the early Christian period population, while absent or found to a lesser extent in the less stressed population, the late Christian period population. We computed two mean values, representative of right-side and left-side epiphyseal union, for each individual in both cemetery samples, and tested for significant differences. Bilateral asymmetry was significant in the combined cemetery sample of 90 individuals (P < 0.019). When cemetery samples were tested separately, bilateral asymmetry was significant for the early Christian period sample (P < 0.001), but not for the late Christian period sample. There were no differences attributable to sex. Finally, we discuss why we conclude that environmental stress was favored over a biomechanic explanation as the cause for asymmetry. To the extent that our results support previous findings that early Christian period individuals were more affected by environmental stress than late Christian period individuals, it is reasonable to consider bilateral asymmetry in skeletal growth and maturation a good indicator of environmental stress.     

兔精子发生过程中cyclin B1基因表达和蛋白定位的发育阶段依赖性

利用原位杂交和免疫组化等方法,研究兔精子发生过程中生精细胞cyclin B1 mRNA的表达和蛋白定位特点,结果显示,兔生精上皮中Cyclin B1 mRNA的主要分布在初级精母细胞中,直至圆形精子细胞仍然存在,于精子细胞的变态过程中逐渐消失,在伸长的精子细胞和精子中未检测出cyclin B1 mRNA,Cyclin B1蛋白在进入分裂期的精原细胞和精母细胞中表达,在圆形精子细胞和伸长的精子细胞中呈现大量的cyclin B1蛋白,上述结果表明,在兔精子发生过程中,cyclin B1 mRNA表达和蛋白定位具有发育阶段依赖性的特征。     

MARCH7 E3 ubiquitin ligase is highly expressed in developing spermatids of rats and its possible involvement in head and tail formation

Spermatogenesis is a highly complicated metamorphosis process of male germ cells. Recent studies have provided evidence that the ubiquitin–proteasome system plays an important role in sperm head shaping, but the underlying mechanism is less understood. In this study, we localized membrane-associated RING-CH (MARCH)7, an E3 ubiquitin ligase, in rat testis. Northern blot analysis showed that March7 mRNA is expressed ubiquitously but highly in the testis and ovary. In situ hybridization of rat testis demonstrated that March7 mRNA is expressed weakly in spermatogonia and its level is gradually increased as they develop. Immunohistochemical analysis detected MARCH7 protein expression in spermiogenic cells from late round spermatids to elongated spermatids and in epididymal spermatozoa. Moreover, MARCH7 was found to be localized to the caudal end of the developing acrosome of late round and elongating spermatids, colocalizing with β-actin, a component of the acroplaxome. In addition, MARCH7 was also detected in the developing flagella and its expression levels were prominent in elongated spermatids. We also showed that MARCH7 catalyzes lysine 48 (K48)-linked ubiquitination. Immunolocalization studies revealed that K48-linked ubiquitin chains were detected in the heads of elongating spermatids and in the acrosome/acroplaxome, neck, midpiece and cytoplasmic lobes of elongated spermatids. These results suggest that MARCH7 is involved in spermiogenesis by regulating the structural and functional integrity of the head and tail of developing spermatids.     

Diadem/crater defects in spermatozoa from two related angus bulls

The diadem/crater defect was studied over several months in two related 20-month-old Angus bulls. In bull 1, diadem/crater defects were present in 2-99% of ejaculated spermatozoa at various times during the evaluation period. In bull 2, affected cells varied from 20% to 94%, with other abnormalities (head and acrosome defects, coiled tails, proximal cytoplasmic droplets) also common. Single sire mating trials conducted over 26 days during an apparent recovery phase showed normal fertility (approximately 50% pregnancies per estrus exposed). Both resting and gonadotropin-releasing hormone (GnRH)-stimulated testosterone values were within nor-mal limits. Histopathological evaluation of testes showed no obvious hypoplastic, inflammatory, or degenerative condition. Electron microscopy of ejaculated spermatozoa demonstrated the characteristic diadem pattern of craters in the equatorial region of the head. Many cells from bull 2 contained large craters in other regions of the nucleus. Electron microscopy of testicular tissue demonstrated nuclear invaginations lined by a single unit membrane in round spermatids. Lesions in elongated spermatids were more pronounced, with curling of the nucleus and large membrane-filled cavities in the chromatin occurring in addition to craters in the equatorial region of the nucleus.     

Differences in early patterns of gonadotrophin secretion between early and late maturing bulls, and changes in semen characteristics at puberty

In prepubertal bull calves there is an early transient rise in gonadotrophin secretion between 10 and 20 wk of age, and it has been suggested that this plays a role in the attainment of sexual maturation. To test this, we looked for differences in the gonadotrophin secretory pattern from birth to puberty between early and late maturing bulls. We also characterized the changes in semen morphology that occur about the time of puberty. Blood samples were collected (n=28) every wk from 2 to 20 wk of age and then every 2 wk until 50 wk of age. Semen was collected by electroejaculation at approximately 4-wk intervals from 36 to 49 wk of age. Puberty was defined as the first age at which an ejaculate contained 50 million spermatozoa with a minimum of 10 % motility Bulls were divided into early (n = 14) and late (n = 14) maturing groups based on the age at puberty (41.9 +/- 0.3 and 48.3 +/- 0.7 wk of age, respectively). There was a transient increase in serum concentrations of LH and FSH between 2 and 24 wk of age; LH concentrations were greater in early maturing bulls than in late maturing bulls at 12, 13, 15, 17 and 48 wk of age (P < 0.05). Serum concentrations of testosterone and FSH did not differ between groups (P > 0.05). As the bulls matured there was an increase in the percentage of normal and live sperm cells, cell motility and the number of cells per ejaculate (P < 0.05), and a decrease in the percentage of proximal droplets and knobbed acrosomes (P < 0.05). We concluded that, during the early rise in LH secretion, early maturing bulls had higher circulating LH concentrations than late maturing bulls. During the weeks preceding and following puberty there was an increase in the quality of semen collected by electroejaculation.     

Restauration de la fertilité masculine par la spermatogenèsein vitro

Anin-vitro culture technique, previously tested with germ cells from men with complete spermatogenesis, was applied in assisted reproduction treatment for cases of meiotic and postmeiotic maturation arrest. Some primary spermatocytes from men with maturation arrest at the pachytene stage developed up to the late elongated spermatid stages and were capable of fertilizing the spouse’s oocytes and of giving rise to embryos that were transferred into the spouse’s uterus and subsequently developed to term. In other cases, round spermatids blocked in vivo before the process of spermiogenesis developed to elongated spermatidsin vitro; with the use of suchin-vitro formed spermatids, the first term pregnancies in cases of complete spermiogenesis arrest were achieved. These findings show that certain in-vivo developmental blocks in male germ cells from patients with severe testiculopathies can be overcome byin-vitro culture, probably by modifying control mechanisms acting at developmental checkpoints.     

Stage-dependent variation in the radiosensitivity of DNA in developing male germ cells

The induction and rejoining of gamma-ray-induced DNA single-strand breaks (SSBs) were measured in the spermatogenic cells of mice using the alkaline elution technique. The animals were injected with [3H]thymidine and sacrificed on subsequent days to examine selectively cohorts of radiolabeled cells in the successive stages of maturation. A significantly increased frequency of SSB was observed in the unirradiated early spermatocytes and late spermatids, associated with genetic recombination and chromatin compaction, respectively. The frequency of SSBs induced by irradiation of animals in vivo remained constant from the early spermatocyte through mid-spermatid stages and decreased significantly only after the cells matured to the late spermatid stage. The frequency of SSBs after in vitro irradiation of testicular cell suspensions also decreased as round spermatids matured to late spermatids. Such decreases for both modes of irradiation may result from maturation-dependent alterations in chromatin in late spermatids, such as condensation and replacement of histones with protamines, rather than from changes in oxygen tension. Rejoining of SSBs in vivo was efficient in the spermatocytes and early spermatids but declined in late spermatids. Possible reasons for the discrepancy between the greater number of unrepaired lesions and lower susceptibility to mutation induction in late spermatids than in round spermatids are discussed.     

Establishment of spermatogenesis in neonatal bovine testicular tissue following ectopic xenografting varies with donor age

Ectopic testicular xenografting can be used to investigate spermatogenesis and as an alternative means for generating transgenic spermatozoa in many species. Improving the efficiency of spermatogenesis in xenografted testicular tissue will aid in the application of using this approach. The present study was conducted to evaluate age-related differences in the establishment of spermatogenesis in grafted testicular tissue from bulls between 2 and 16 wk of life. Testicular tissue was ectopically xenografted under the skin on the backs of castrated nude mice and subsequently evaluated for growth, testosterone production, and establishment of spermatogenesis 24 wk after grafting. The greatest weight increases occurred in donor tissue from calves of the ages 2, 4, and 8 wk compared with the ages of 12 and 16 wk. Recipient mouse serum testosterone concentration was at normal physiological levels 24 wk after grafting and no significant differences were detected between recipients grafted with testicular tissue from bull calves of different ages. The development of germ cells to elongated spermatids were observed in seminiferous tubules of grafts from donor calves of the ages 4, 8, 12, and 16 wk but not observed in grafts from 2-wk donors, which contained round spermatids as the most advanced germ cell stage. Grafts from 8-wk donors contained a significantly higher (10-fold) average percentage of seminiferous tubules with elongated spermatids than all other donor ages. These data demonstrate differences in the ability of testicular tissue from donor animals of different ages to establish spermatogenesis following ectopic testicular xenografting.