Adult Sertoli cell differentiation in settings of normal and impaired spermatogenesis

Successful spermatogenesis during adult life is dependent upon the establishment of a normal complement of fully differentiated Sertoli cells, which occurs around puberty. The transition from an undifferentiated to a differentiated Sertoli cell population involves a series of structural, functional and regulatory changes that are essential to support and cater for the individual needs of each germ cell type. For over a century it has been believed that once adult Sertoli cells complete differentiation that their differentiation state is fixed for life, a feature called 'terminal' differentiation. However, research efforts over the past decade largely from the seasonal-breeding Djungarian hamster and humans reveal that adult Sertoli cells are capable of dedifferentiating in settings of impaired spermatogenesis, and therefore may not be a homogenous 'terminally' differentiated population. These adult Sertoli cells feature an 'intermediate' state of differentiation, in that they display at least one hallmark characteristic of a prepubertal undifferentiated Sertoli cell. Whilst the hamster model has been invaluable, to advance our understanding of adult Sertoli cell differentiation and its impact on spermatogenesis, access to traditional laboratory species that feature more similar breeding systems to humans, such as the rat and mouse, and clinical samples, are needed.

This thesis hypothesised that adult rodent Sertoli cells are not 'terminally' differentiated in settings of normal and impaired spermatogenesis in vivo, and that the extent to which adult Sertoli cells are differentiated in settings of impaired spermatogenesis is linked to the level of spermatogenic impairment.

This thesis extends our understanding of in vivo adult Sertoli cell differentiation by addressing three key aims:
i) To establish whether adult Sertoli cells from laboratory animals are 'terminally' differentiated (Chapters 3 & 4).
ii) To assess the relationship between adult Sertoli cell differentiation status and germ cell progression (Chapters 2 & 3).
iii) To determine the extent to which adult Sertoli cells are differentiated in settings of normal and impaired spermatogenesis through examination of specific structural, functional and/or regulatory changes (Chapters 2, 3 & 4).

To do this, a combination of novel approaches using clinical tissues and/or laboratory animal models featuring normal and impaired spermatogenesis were applied throughout this thesis to examine broadly three hallmark characteristics of Sertoli cell differentiation; Sertoli cell junction organisation (Chapter 2), blood-testis barrier (BTB) function (Chapter 3) and Sertoli cell proliferation (Chapter 4).

Chapter 2: Limited evidence suggests human adult Sertoli cells are not 'terminally' differentiated in vivo. In healthy men with suppressed spermatogenesis, adult Sertoli cells exhibit proliferative potential (Tarulli et al., 2013), and in men with idiopathic impaired spermatogenesis, BTB function is compromised (Cavicchia et al., 1996). This chapter further clarifies and quantifies the extent to which adult Sertoli cells are differentiated in clinical tissues featuring impaired spermatogenesis and its association with germ cell progression, using Sertoli cell junction organisation as a marker of differentiation status. To do this, a new approach to measure morphologic changes in Sertoli cell tight and gap junction organisation was developed. Four distinct morphological patterns of claudin-11 and connexin-43 organisation were identified, and quantitative analysis revealed significant (P<0.001) differences in the extent of organisation of these junctional proteins in tubules with impaired spermatogenesis compared with qualitatively normal spermatogenesis. However, interestingly there was no difference in the extent of Sertoli cell differentiation, based on junctional criteria, in tubules with no germ cells compared to those with germ cells present up to spermatocytes. This chapter provides the first detailed molecular account on the organisation of Sertoli cell junction proteins at the human blood-testis barrier (BTB). It is concluded, based on junctional criteria as a marker of Sertoli cell differentiation, that there is no link between Sertoli cell differentiation status and germ cell progression in settings of idiopathic impaired spermatogenesis in men.

Chapter 3: Evidence from the Djungarian hamster (Tarulli et al., 2006, 2008) and adult rat (McCabe et al., 2010) show that adult BTB function is compromised in settings of impaired spermatogenesis, suggesting that adult rat Sertoli cells can dedifferentiate. This chapter dissects out the extent of adult Sertoli cell dedifferentiation and its association with germ cell progression, using BTB function as a marker. To do this, a new approach to measure the extent of BTB function was developed in an adult rat model of spermatogenic impairment and reinitiation using three tracers of small (0.6 kDa), medium (70 kDa) and large (150 kDa) molecular weights (MWs), to emulate the size of blood- and lymphatic-borne factors that could cross the BTB. It was found that the BTB is selectively permeable to MWs up to 70 kDa, but not 150 kDa, and that this permeability relates to the presence of particular meiotic and post-meiotic germ cells in the epithelium. The results of this chapter reveal for the first time that the BTB features differing functionality, and challenges the current model of BTB function as being either ‘open’ or ‘closed’. It is concluded, based on BTB function as a marker of Sertoli cell differentiation, that there is a relationship between Sertoli differentiation status and germ cell progression during spermatogenic reinitiation in healthy adult rats.

Chapter 4: Recent stereological evidence reveals that adult Sertoli cell number increases beyond puberty in normal mice (Hazra et al., 2013), implying that adult mouse Sertoli cells may retain the ability to proliferate. This chapter first sought to assess the differentiation status of normal mouse adult Sertoli cells, using proliferation as a marker. It was found that approximately 3% of normal adult mouse Sertoli cells exhibit proliferative activity, revealing the suitability of this laboratory animal as a new model to study adult Sertoli cell differentiation. It is well established that FSH can regulate Sertoli cell differentiation, at least in the Djungarian hamster, based on extensive proliferation assessments (Meachem et al., 2005a; Tarulli et al., 2006). Therefore, this chapter also explored the role of FSH action during postnatal and postpubertal life on adult Sertoli cell differentiation status using proliferation as a marker in the inhibin alpha knockout mouse. This model is both particularly sensitive to FSH action and features abnormally sustained Sertoli cell proliferation during adulthood resulting in Sertoli cell tumours. The results show that FSH inhibition during only one week of postnatal or postpubertal life suppresses the abnormal adult Sertoli cell proliferation in inha KO mice to near normal levels, subsequently suppressing tumour growth. It is concluded, based on proliferation as a marker of Sertoli cell differentiation, that the differentiation status of adult Sertoli cells is regulated by FSH action during both postnatal and postpubertal life.

This thesis has added four important pieces of information to the field of adult Sertoli cell differentiation:
i) Sertoli cell junctional proteins are disorganised at the adult inter-Sertoli interface in men with idiopathic impaired spermatogenesis, and the extent of junction disorganisation is not related to the extent of germ cell progression.
ii) The BTB is not just 'open' and 'shut' in healthy adult rats; it features differing levels of functionality that are related to the extent of germ cell progression.
iii) A subset of normal adult mouse Sertoli cells exhibit proliferative activity. iv) FSH regulates adult mouse Sertoli cell differentiation, at least in terms of proliferation.

Additionally, this thesis has also made three important technical contributions to the field of Sertoli cell differentiation:
i) Identifying four descriptors of human adult Sertoli cell junction organisation.
ii) Development of a more comprehensive approach to test BTB functionality.
iii) Description of two readily accessible laboratory animals as suitable models to further study Sertoli cell differentiation, an important area that has been under researched due to restricted access to well characterised models such as the Djungarian hamster.

In conclusion, human, rat and mouse adult Sertoli cells are not a homogenous 'terminally' differentiated population, but rather are a heterogeneous population that feature an 'intermediate' state of differentiation in settings of normal and impaired spermatogenesis. These 'intermediate' Sertoli cells can arise from either dedifferentiation during adult life (Chapter 3) or may be cells that never reach their fully differentiated form during postnatal and pubertal development, due either to unknown genetic factors (Chapter 2) or in response to a changing hormonal environment (Chapter 4). These findings reveal a deeper understanding of adult Sertoli cell plasticity, and highlight the importance of Sertoli cell differentiation for complete germ cell progression.