SALL4 Expression in Gonocytes and Spermatogonial Clones of Postnatal Mouse Testes

The spermatogenic lineage is established after birth when gonocytes migrate to the basement membrane of seminiferous tubules and give rise to spermatogonial stem cells (SSC). In adults, SSCs reside within the population of undifferentiated spermatogonia (Aundiff) that expands clonally from single cells (Asingle) to form pairs (Apaired) and chains of 4, 8 and 16 Aaligned spermatogonia. Although stem cell activity is thought to reside in the population of Asingle spermatogonia, new research suggests that clone size alone does not define the stem cell pool. The mechanisms that regulate self-renewal and differentiation fate decisions are poorly understood due to limited availability of experimental tools that distinguish the products of those fate decisions. The pluripotency factor SALL4 (sal-like protein 4) is implicated in stem cell maintenance and patterning in many organs during embryonic development, but expression becomes restricted to the gonads after birth. We analyzed the expression of SALL4 in the mouse testis during the first weeks after birth and in adult seminiferous tubules. In newborn mice, the isoform SALL4B is expressed in quiescent gonocytes at postnatal day 0 (PND0) and SALL4A is upregulated at PND7 when gonocytes have colonized the basement membrane and given rise to spermatogonia. During steady-state spermatogenesis in adult testes, SALL4 expression overlapped substantially with PLZF and LIN28 in Asingle, Apaired and Aaligned spermatogonia and therefore appears to be a marker of undifferentiated spermatogonia in mice. In contrast, co-expression of SALL4 with GFRα1 and cKIT identified distinct subpopulations of Aundiff in all clone sizes that might provide clues about SSC regulation. Collectively, these results indicate that 1) SALL4 isoforms are differentially expressed at the initiation of spermatogenesis, 2) SALL4 is expressed in undifferentiated spermatogonia in adult testes and 3) SALL4 co-staining with GFRα1 and cKIT reveals distinct subpopulations of Aundiff spermatogonia that merit further investigation. © 2013 Gassei, Orwig

Measuring Behavior in the Home Cage : Study Design, Applications, Challenges, and Perspectives

FUNDING This research was supported by NIH grants R00AG056662, P20GM125528, AG057424 to WS and SLPeer reviewedPublisher PD

A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse

In whole mounts of seminiferous tubules of C3H/101 F1 hybrid mice, spermatogonia were counted in various stages of the epithelial cycle. Furthermore, the total number of Sertoli cells per testis was estimated using the disector method. Subsequently, estimates were made of the total numbers of the different spermatogonial cell populations per testis. The results of the cell counts indicate that the undifferentiated spermatogonia are actively proliferating from stage XI until stage IV. Three divisions of the undifferentiated spermatogonia are needed to obtain the number of A1 plus undifferentiated spermatogonia produced each epithelial cycle. Around stage VIII almost two-thirds of the Apr and all of the Aal spermatogonia differentiate into A1 spermatogonia. It was estimated that there are 2.5 x 10(6) differentiating spermatogonia and 3.3 x 10(5) undifferentiated spermatogonia per testis. There are about 35,000 stem cells per testis, constituting about 0.03% of all germ cells in the testis. It is concluded that the undifferentiated spermatogonia, including the stem cells, actively proliferate during about 50% of the epithelial cycl

Chapter 30 - Software tools for behavioral phenotyping of zebrafish across the life span

In this chapter, we address how one can detect and measure specific behaviors in zebrafish, from the embryonic stage to adulthood, and in various behavioral paradigms using automated, image-based analysis software. In the first section, we address the measurement of activity in embryos and heart rate in larvae. For the larval stage, we also show how software can analyze the fish's body shape and movement trajectory to measure activity and specific behaviors like avoidance and escape responses. Custom output variables address specific research questions and increase throughput. For adult zebrafish, we describe applications with the novel tank diving test, learning in the T-maze, aggressive behavior in mirror tests, and quantify shoaling behavior. The advantages of tracking in three dimensions relative to classic 2D tracking are also addressed. Finally, we discuss current limitations and directions for future development

An automated system for the recognition of various specific rat behaviours

The automated measurement of rodent behaviour is crucial to advance research in neuroscience and pharmacology. Rats and mice are used as models for human diseases; their behaviour is studied to discover and develop new drugs for psychiatric and neurological disorders and to establish the effect of genetic variation on behavioural changes. Such behaviour is primarily labelled by humans. Manual annotation is labour intensive, error-prone and subject to individual interpretation. We present a system for automated behaviour recognition (ABR) that recognises the rat behaviours ‘drink’, ‘eat’, ‘sniff’, ‘groom’, ‘jump’, ‘rear unsupported’, ‘rear wall’, ‘rest’, ‘twitch’ and ‘walk’. The ABR system needs no on-site training; the only inputs needed are the sizes of the cage and the animal. This is a major advantage over other systems that need to be trained with hand-labelled data before they can be used in a new experimental setup. Furthermore, ABR uses an overhead camera view, which is more practical in lab situations and facilitates high-throughput testing more easily than a side-view setup. ABR has been validated by comparison with manual behavioural scoring by an expert. For this, animals were treated with two types of psychopharmaca: a stimulant drug (Amphetamine) and a sedative drug (Diazepam). The effects of drug treatment on certain behavioural categories were measured and compared for both analysis methods. Statistical analysis showed that ABR found similar behavioural effects as the human observer. We conclude that our ABR system represents a significant step forward in the automated observation of rodent behaviour

An automated system for the recognition of various specific rat behaviours

The automated measurement of rodent behaviour is crucial to advance research in neuroscience and pharmacology. Rats and mice are used as models for human diseases; their behaviour is studied to discover and develop new drugs for psychiatric and neurological disorders and to establish the effect of genetic variation on behavioural changes. Such behaviour is primarily labelled by humans. Manual annotation is labour intensive, error-prone and subject to individual interpretation. We present a system for automated behaviour recognition (ABR) that recognises the rat behaviours ‘drink’, ‘eat’, ‘sniff’, ‘groom’, ‘jump’, ‘rear unsupported’, ‘rear wall’, ‘rest’, ‘twitch’ and ‘walk’. The ABR system needs no on-site training; the only inputs needed are the sizes of the cage and the animal. This is a major advantage over other systems that need to be trained with hand-labelled data before they can be used in a new experimental setup. Furthermore, ABR uses an overhead camera view, which is more practical in lab situations and facilitates high-throughput testing more easily than a side-view setup. ABR has been validated by comparison with manual behavioural scoring by an expert. For this, animals were treated with two types of psychopharmaca: a stimulant drug (Amphetamine) and a sedative drug (Diazepam). The effects of drug treatment on certain behavioural categories were measured and compared for both analysis methods. Statistical analysis showed that ABR found similar behavioural effects as the human observer. We conclude that our ABR system represents a significant step forward in the automated observation of rodent behaviour