Dickkopf homolog 3 (DKK3) plays a crucial role upstream of WNT/β-CATENIN signaling for sertoli cell mediated regulation of spermatogenesis

Testicular Sertoli cells (Sc) are main somatic component of seminiferous tubules that govern the differentiation of germ cells (Gc) and provide them physical support. Sc are the target of follicle stimulating hormone (FSH) and testosterone (T) which are known to regulate spermatogenesis. FSH and T levels in human and sub-human male primates remain high during infancy (4–6 months post birth), similar to those during puberty. Subsequently, juvenile phase is marked with low levels of these hormones. In spite of prolonged hormonal exposure, spermatogenesis is not discerned during infancy unlike that during puberty. Situation during infancy is similar to certain idiopathic male infertility, where prolonged hormone supplementation fails to initiate spermatogenesis. In our quest to determine non hormonal causes of idiopathic infertility which may reside within the Sc, we investigated the association between spermatogenesis and Sc specific gene(s) expressed differentially during puberty and infancy. Although products of several genes may be necessary for quantitatively normal spermatogenesis, one needs to investigate their roles one by one. Differential display and real time PCR analysis revealed higher expression of a known tumor suppressor, Dickkopf homolog 3 (DKK3), by pubertal monkey Sc as compared to infant Sc. To evaluate role of DKK3 in spermatogenesis, we generated DKK3 knock down mice (DKDM) using shRNA construct targeted to DKK3. In testis of adult DKDM, expression of DKK3 mRNA and protein were significantly (p<0.05) low and was associated with elevated WNT-4/β-CATENIN activity. Elevated β-CATENIN activity is known to restrict Sc maturation. Abundant expression of infant Sc marker, Mullerian inhibiting substance (MIS), in the testes of adult DKDM confirmed lack of Sc maturation in DKDM. Gc differentiation and fertility was severely compromised in DKDM. This is the first report of role of DKK3 in the testis and DKK3 mediated regulation of spermatogenesis via WNT-4/β-CATENIN modulation

An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation

Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis

An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation.

Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis

An efficient method for generation of transgenic rats avoiding embryo manipulation

Although rats are preferred over mice as an animal model, transgenic animals are generated predominantly using mouse embryos. There are limitations in the generation of transgenic rat by embryo manipulation. Unlike mouse embryos, most of the rat embryos do not survive after male pronuclear DNA injection which reduces the efficiency of generation of transgenic rat by this method. More importantly, this method requires hundreds of eggs collected by killing several females for insertion of transgene to generate transgenic rat. To this end, we developed a noninvasive and deathless technique for generation of transgenic rats by integrating transgene into the genome of the spermatogonial cells by testicular injection of DNA followed by electroporation. After standardization of this technique using EGFP as a transgene, a transgenic disease model displaying alpha thalassemia was successfully generated using rats. This efficient method will ease the generation of transgenic rats without killing the lives of rats while simultaneously reducing the number of rats used for generation of transgenic animal

A switch in Sertoli cell responsiveness to FSH may be responsible for robust onset of germ cell differentiation during prepubartal testicular maturation in rats

FSH and Testosterone (T) regulate spermatogenesis via testicular Sertoli cells (Sc), which bear receptors for these hormones. Despite sufficient circulating levels of FSH and T postnatally, predominant appearance of spermatogonia B and spermatocytes is not discernible until 11 and 18 days of postnatal age, respectively, in rat testes. In an attempt to explore the underlying causes, we cultured Sc from neonatal (5- and 9-day-old) and prepubertal (12- and 19-day-old) rat testes and compared the status of FSH receptor (FSH-R) and androgen receptor (AR) signaling. Protein and mRNA levels of FSH-R and AR remained uniform in cultured Sc from all age groups. Androgen binding ability of AR was similar, and T-induced nuclear localization of AR was discernible in Sc from all age groups. Binding of FSH to FSH-R, subsequent production of cAMP, and mRNA of stem cell factor (SCF) and glial cell line-derived neurotrophic factor (GDNF), known to be essential for the robust differentiation of repopulating spermatogonia, were significantly augmented in prepubertal Sc compared with those in neonatal Sc. However, treatment of neonatal Sc with cholera toxin or forskolin, which stimulate cAMP production bypassing FSH-R, demonstrated a concomitant rise in SCF and GDNF mRNA expression, which was similar to the FSH-mediated rise observed in prepubertal Sc. These observations suggested that, during prepubertal Sc maturation, the ability of FSH-R to respond to FSH is significantly augmented and is associated with the robust differentiation of repopulating spermatogonia, and such a switch in Sc from FSH-resistant to FSH-responsive mode during prepubertal development may underlie the initiation of robust spermatogenesis

FSH mediated cAMP signalling upregulates the expression of Gα subunits in pubertal rat Sertoli cells

Follicle Stimulating Hormone (FSH) acts via FSH-Receptor (FSH-R) by employing cAMP as the dominant secondary messenger in testicular Sertoli cells (Sc) to support spermatogenesis. Binding of FSH to FSH-R, results the recruitment of the intracellular GTP binding proteins, either stimulatory Gαs or inhibitory Gαi that in turn regulate cAMP production in Sc. The cytosolic concentration of cAMP being generated by FSH-R thereafter critically determines the downstream fate of the FSH signalling. The pleiotropic action of FSH due to differential cAMP output during functional maturation of Sc has been well studied. However, the developmental and cellular regulation of the Gα proteins associated with FSH-R is poorly understood in Sc. In the present study, we report the differential transcriptional modulation of the Gα subunit genes by FSH mediated cAMP signalling in neonatal and pubertal rat Sc. Our data suggested that unlike in neonatal Sc, both the basal and FSH/forskolin induced expression of Gαs, Gαi-1, Gαi-2 and Gαi-3 transcripts was significantly (p < 0.05) up-regulated in pubertal Sc. Further investigations involving treatment of Sc with selective Gαi inhibitor pertussis toxin, confirmed the elevated expression of Gi subunits in pubertal Sc. Collectively our results indicated that the high level of Gαi subunits serves as a negative regulator to optimize cAMP production in pubertal Sc

Cortactin interacts with αDystrobrevin-1 and regulates murine neuromuscular junction morphology

Neuromuscular junctions transmit signals from the nervous system to skeletal muscles, triggering their contraction, and their proper organization is essential for breathing and voluntary movements. αDystrobrevin-1 is a cytoplasmic component of the dystrophin-glycoprotein complex and has pivotal functions in regulating the integrity of muscle fibers and neuromuscular junctions. Previous studies identified that αDystrobrevin-1 functions in the organization of the neuromuscular junction and that its phosphorylation in the C-terminus is required in this process. Our proteomic screen identified several putative αDystrobrevin-1 interactors recruited to the Y730 site in phosphorylated and unphosphorylated states. Amongst various actin-modulating proteins, we identified the Arp2/3 complex regulator cortactin. We showed that similarly to αDystrobrevin-1, cortactin is strongly enriched at the neuromuscular postsynaptic machinery and obtained results suggesting that these two proteins interact in cell homogenates and at the neuromuscular junctions. Analysis of synaptic morphology in cortactin knockout mice showed abnormalities in the slow-twitching soleus muscle and not in the fast-twitching tibialis anterior. However, muscle strength examination did not reveal apparent deficits in knockout animals

Low levels of Gαs and Ric8b in testicular sertoli cells may underlie restricted FSH action during infancy in primates

FSH acts via testicular Sertoli cells (Sc) bearing FSH receptor (FSH-R) for regulating male fertility. Despite an adult-like FSH milieu in infant boys and monkeys, spermatogenesis is not initiated until the onset of puberty. We used infant and pubertal monkey Sc to reveal the molecular basis underlying developmental differences of FSH-R signaling in them. Unlike pubertal Sc, increasing doses of FSH failed to augment cAMP production by infant Sc. The expression of Gαs subunit and Ric8b, which collectively activate adenylyl cyclase (AC) for augmenting cAMP production and gene transcription, were significantly low in infant Sc. However, forskolin, which acts directly on AC bypassing FSH-R, augmented cAMP production and gene transcription uniformly in both infant and pubertal Sc. FSH-induced Gαs mRNA expression was higher in pubertal Sc. However, Gαi-2 expression was down-regulated by FSH in pubertal Sc, unlike infant Sc. FSH failed, but forskolin or 8-Bromoadenosine 3',5'-cyclic monophosphate treatment to infant Sc significantly augmented the expression of transferrin, androgen binding protein, inhibin-β-B, stem cell factor, and glial-derived neurotropic factor, which are usually up-regulated by FSH in pubertal Sc during spermatogenic onset. This suggested that lack of FSH mediated down-regulation of Gαi-2 expression and limited expression of Gαs subunit as well as Ric8b may underlie limited FSH responsiveness of Sc during infancy. This study also divulged that intracellular signaling events downstream of FSH-R are in place and can be activated exogenously in infant Sc. Additionally, this information may help in the proper diagnosis and treatment of infertile individuals having abnormal G protein-coupled FSH-R

<i>DKK3</i> is over expressed in Sc during puberty of monkeys and mice.

<p>(A) Testicular sections showing seminiferous tubules of an infant monkey. Spermatogonia A are shown with arrow. Scale bar: 50 µm (B) Testicular sections showing seminiferous tubules of pubertal monkey. Spermatogonia B (shown with arrow) and spermatocytes (shown with arrowhead) can be noticed in pubertal monkey. Scale bar: 50 µm (C) Relative quantity of <i>DKK3</i> mRNA expression in Sc from infant (open bar) and pubertal monkeys (hatched bar). Real time PCR data from Sc of three animals are represented as mean +/− SEM in each bar (*p<0.05). (D) Relative quantity of <i>DKK3</i> expression in Sc from 7 days old (open bar) and 20 days old mice (hatched bar). Real time PCR data from Sc of three animals are represented as mean +/− SEM in each bar (*p<0.05).</p