Early Phases Of Spermatogenesis Research Articles

Down-Regulation of Neogenin Decreases Proliferation and Differentiation of Spermatogonia during the Early Phase of Spermatogenesis

Non-obstructive azoospermia is a major clinical issue associated with male infertility that remains to be addressed. Although neogenin is reportedly abundantly expressed in the testis, its role in mammalian spermatogenesis is unknown. We systematically investigated the role of neogenin during spermatogenesis by performing loss-of-function studies. Testis-specific neogenin conditional knock-out (cKO) mice were generated using CRISPR/Cas9 and neogenin-targeting guide RNAs. We analyzed the expression profiles of germ cell factors by RT-PCR and Western blotting. Neogenin localized mainly to spermatogonia in seminiferous tubules of mouse testes. RT-PCR and Western blot analyses further demonstrated that neogenin expression varied during spermatogenesis and was dramatically increased at postnatal day 12-25 during the pubertal stage. In neogenin-cKO mouse testes, the ratio of primary and secondary spermatocytes was significantly decreased compared with the control, while the number of apoptotic testicular cells was significantly increased. Taken together, these results suggest that neogenin plays a pivotal role in the maintenance and proliferation of spermatogonia during the early stage of spermatogenesis in mice.

Proteomic analysis of pikeperch seminal plasma provides novel insight into the testicular development of domesticated fish stocks

Control of the reproduction of domesticated stocks is considered a prerequisite for aquaculture development of pikeperch. However, knowledge about the physiology of the captive pikeperch male reproductive system and the biology of semen is very limited, especially regarding protein characteristics. The aims of our study were to characterize pikeperch sperm quantity and quality parameters and to analyze changes in the proteome of the same males spawned for the first and second times. Moreover, attempts were made to generate the first proteomic library of seminal plasma proteins. Semen collected during the first spawning season was characterized by lower sperm concentration and volume than for the second season. Using mass spectrometry-based label-free quantitative proteomics, we identified 850 proteins in the seminal plasma of pikeperch from both spawning seasons, and 65 seminal proteins were found to be differentially abundant between the first and second spawning seasons. The majority of differentially abundant proteins were involved in stress and immune responses, developmental processes, cofactor metabolic processes, proteolysis, cellular oxidant detoxification and organization of the extracellular matrix (ECM). In addition, several proteins unique to pikeperch seminal plasma were identified, including antifreeze proteins, hibernation-specific plasma proteins, lectins and vitellogenin. In summary, our results indicate that males that spawned for the first time were characterized by incompletely mature gonads and the expression of proteins associated with the early phase of spermatogenesis and ECM organization. On the other hand, males that spawned for the second time exhibited advanced gonadal maturation and expression of proteins related to the late stage of spermatogenesis and sperm maturation, including regulation of reactive oxygen species generation, bicarbonate production, sperm elongation and separation. The identification of a large number of seminal plasma proteins provides a valuable resource for understanding the functions of seminal plasma and the molecular mechanisms involved in testicular development and maturation in domesticated fish, which is a prerequisite for better control of reproduction in captivity.

Particulate Pollution and Male Reproductive Health: Differential Impacts in Early and Late Spermatogenesis

Particulate Pollution and Male Reproductive Health: Differential Impacts in Early and Late SpermatogenesisBackground/Aim: Despite increasing evidence that particulate air pollution has adverse effects on human semen quality, few studies examine clinically relevant thresholds typically employed by clinicians to diagnose male fertility problems. Furthermore, exposure is often assessed using average air pollution levels in a geographic area rather than individualized measures. Finally, physiology-driven selection exposure windows are inconsistent.Methods: We examined a previous cohort of 132 healthy males seeking to become parents, using spermatogenesis-relevant exposure windows of 77-34 days and 37-0 days prior to semen collection. Exposure to PM2.5 and PM10 was individualized by selecting multiple air pollution sensors within participants’ geographic air basins and employing inverse distance weighting to calculate mean daily exposure levels. We used multiple logistic regression to assess the association between pollution, temperature, and dichotomized World Health Organization semen parameters.Results: During the early phase of spermatogenesis, air pollution exposure is associated with 1.563 (95% CI, 1.049 to 2.452) times greater odds of <30% normal heads per 1-unit increase in IQR for PM2.5. In the late phase of spermatogenesis, air pollution exposure is associated with 0.368 (95% CI, 0.114 to 0.767) times greater odds of semen concentration <15 million/mL per 1-unit increase in IQR for PM2.5, and 0.208 (95% CI, 0.042 to 0.653) for PM10.Conclusions: Particulate exposure has a differential and more deleterious impact on early-phase spermatogenesis than late-phase, especially for PM2.5.This suggests that smaller particles breathed earlier in the spermatogenic cycle are likelier to affect sperm head formation.

HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse.

piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2’ O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program.

Zygotic chromosomal structural aberrations after paternal drug treatment.

In recent years, the field of male-mediated reproductive toxicology has received growing attention. It is now well-established that many drugs, chemicals, and environmental factors can harm male germ cells by inducing DNA damage. Male germ cells have extensive repair mechanisms that allow detection and repair of damaged DNA during the early phases of spermatogenesis. However, during the later phase of spermiogenesis, when the haploid spermatids undergo chromatin condensation and become transcriptionally quiescent, their ability to repair damaged DNA is lost.12 It is also thought that the highly compacted chromatin of the sperm can protect DNA against damage.3 Therefore, it is expected that late spermatids will be most susceptible to DNA damaging agents. Unrepaired or misrepaired damage in the germ cells leads to the generation of spermatozoa with DNA damage that can be transmitted to the next generation. Fortunately, the maternal DNA repair machinery is capable of recognizing and repairing, at least to some degree, damaged paternal DNA after fertilization in the zygote. Therefore, the efficiency of the maternal repair machinery will greatly influence the risk of transmitting paternal DNA damage to offspring.4

Microtubule Configuration and Its Relationship to Sperm Morphology in Mammalian Spermiogenesis

The acrosome is a secretory vesicle located in the mammalian sperm head. Its main function is to transport hydrolytic enzymes. These enzymes will dissolve the zona pellucid, assisting the sperm in fertilizing the egg. The acrosome is assembled in the haploid spermatid during spermiogenesis, but many of its enzymes are already synthesized in pachytene spermatocytes during the early phase of spermatogenesis. Haploid spermatids have developed unique mechanism(s) to assure the proper localization/orientation of the acrosome, attachment near the nucleus, and the targeting of acrosomal proteins towards this vesicle. During spermiogenesis, these germ cells undergo dramatic transformations in shape and intracellular distribution of organelles, and the configuration of microtubules seems to be involved with each specific step. In this context, it seems microtubules are essential for the assembly and formation of the acrosome during spermiogenesis. Microtubules are also involved in guiding proteins to the acrosome, since genetic mouse models with abnormal spermatozoa as their only phenotype were shown to have alterations of the microtubule configuration in round and elongating spermatids, while their spermatozoa were having misplacement of acrosomal proteins. A few genetic mouse models have been designed to study vesicular trafficking or acrosome assembly, and some of these models closely resemble specific human fertility pathologies such as oligoasthenospermia or acrosomeless round-headed sperm. Thus, the study of acrosome biogenesis could aid the development of new infertility tests or the discovery of new regulation targets of sperm production. Keywords: Spermatogenesis, cytoskeleton, spermatid, acrosome

Some steps of spermatogenesis in Halichondria semitubulosa (Demospongiae, Halichondriidae)

Spermatogenesis of the marine sponge Halichondria semitubulosa (formerly Pellina semitubulosa) has been investigated at the ultrastructural level. This process can be observed in March when, among the choanocyte chambers of the aquiferous system, spermatic cysts are visible. They are delimited by pinacocyte‐like cells and include elements in progressive development: spermatocytes of the first and second order, spermatids and spermatozoa. The early phase of spermatogenesis was not detected. Spermatocytes of the first order show an elongated shape, several small mitochondria and patched chromatin; spermatocytes of the second order, frequently connected by bridges, show denser chromatin, a single large mitochondrion with numerous tightly adherent cristae, glycogen, and round‐shaped inclusions with a central electron‐dense core. In the spermatids the chromatin tends to be packed in the central region. Spermatozoa have a uniformly dense nucleus in close association with the large mitochondrion. Sperm maturation takes place synchronously within the same cyst but asynchronously within the same specimen.

Developmental stage-specific expression of Rbm suggests its involvement in early phases of spermatogenesis

Developmental stage-specific expression of Rbm suggests its involvement in early phases of spermatogenesis

Developmental stage-specific expression of Rbm suggests its involvement in early phases of spermatogenesis

Rbm is a male infertility gene located on the Y chromosome that is expressed in the testis. To investigate the specific events of spermatogenesis in which Rbm plays a role, the precise pattern of expression of Rbm in the mouse testis was determined. An antibody was generated against the Rbm protein and used to detect a single specific band of 43 kDa in size in mouse testicular lysates. In situ hybridization, immunoblot and immunohistochemistry analyses together indicated that Rbm was expressed in spermatogonia, preleptotene spermatocytes, late leptotene to early pachytene spermatocytes but not in mid-pachytene spermatocytes or subsequent stages of differentiation, including haploid germ cells. These observations suggest that Rbm functions in early but not later stages of male germ cell development.

Human tumor rejection antigens MAGE.

Characterization of the MAGE genes has facilitated a molecular approach to identification of the genes encoding tumor-rejection antigens expressed on human cancer cells. MAGE proteins are normal tissue antigens compartmentalized in testicular cells that play an important role in the early phase of spermatogenesis. MAGE-1, -2, -3, -4, and -6 genes are preferentially expressed in many different cancers at both the mRNA and protein levels. More than half of human cancers of various histologic type express at least one of these MAGE genes. Demethylation induces MAGE antigens in cells, suggesting that MAGE genes are important developmentally regulated genes under methylating control. Thus, genetic instability in cells causing loss of this methylating control could result in the preferential expression of MAGE genes in cancer cells. Therefore, MAGE gene products may be appropriate target molecules for development of new cancer vaccine.

Effects of hypophysectomy on structure and function of testis in adult toads, Bufo bufo bufo (L.)

The pars distalis of the pituitary gland of sexually mature toads was extirpated, and the effects of this on spermatogenesis and the histological appearance of the interstitial tissue of the testis were studied. The rates of cell multiplication in the early phases of spermatogenesis were estimated from the frequency at which spermatocysts became labeled after a single injection of [ 3H]thymidine. In one experiment, each toad was injected six times with 100 IU of human chorionic gonadotropin (hCG) prior to hypophysectomy. This treatment induced spermiation and cleared the seminiferous tubules of sperm bundles. The treatment also induced a spermatogenetic wave, which reached the sperm bundle stage within 5 to 6 weeks. The pretreatment with hCG further stimulated the interstitial tissue to hypertrophic and hyperplastic growth. After hypophysectomy the interstitial tissue regressed, and it became atrophic within 9 to 10 weeks. In the intact toads, the interstitial tissue underwent partial regression that became maximal about 5–6 weeks after discontinuation of the hCG treatment; the tissue then returned to a fully active appearance. This temporary regression of the interstitial tissue in intact toads may be causally related to pretreatment with high doses of hCG. Hypophysectomy had no measurable effect on spermatogenesis from the stage of primary spermatocytes and onward, whereas production and survival of secondary spermatogonial cysts were strongly impaired. Hypophysectomy also reduced the rate of multiplication of primary spermatogonia, but the effect was delayed and less severe than that on secondary spermatogonia. It was shown that gonadotropin independence during the process of spermatogenesis in the toad is acquired late in the secondary spermatogonial phase. With about 2 months of hypophysectomy the seminiferous tubules began to disintegrate and became infiltrated with connective tissue.

Changes in plasma testosterone levels during the annual reproductive cycle of the hibernating bat, Myotis lucifugus lucifugus with a survey of plasma testosterone levels in adult male vertebrates.

Plasma testosterone concentrations were measured by radioimmunoassay in a wild population of the male hibernating bat, Myotis lucifugus lucifugus, (Chiroptera;Vespertilionidae) throughout the entire annual reproductive cycle. Baseline levels (2.5 ± 0.3-11.7 ± 3.2 ng/ml) occur during the early phases of spermatogenesis (May to mid-July), during the breeding period (September to May) and throughout hibernation (late September to April). These levels are similar to those reported for most adult breeding mammals. During the latter part of spermatogenesis (mid-July to late August) and just prior to maximum hypertrophy of the sex accessory glands, plasma testosterone rises to very high levels with a peak (59.1 ± 9.2 ng/ml) occurring in mid-August. This peak is one of the highest known values for adult mammals. The data suggest that a testosterone surge is required to stimulate the accessory glands fully, but lower levels of androgen are able to support them (as well as epididymal sperm and libido) during the breeding and hibernation seasons. However, these “lower” levels apparently are not sufficient to maintain the accessories when the bats emerge from hibernation in the spring, because these glands involute soon thereafter.