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 efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation

Background: Spermatogonial stem cell (SSC) transplantation (SSCT) has become important for conservation of endangered species, transgenesis and for rejuvenating testes which have lost germ cells (Gc) due to gonadotoxic chemotherapy or radiotherapy during the prepubertal phase of life. Creating a germ cell-depleted animal model for transplantation of normal or gene-transfected SSC is a prerequisite for such experimental studies. Traditionally used intraperitoneal injections of busulfan to achieve this are associated with painful hematopoietic toxicity and affects the wellbeing of the animals. Use of testicular busulfan has been reported recently to avoid this but with a very low success rate of SSCT. Therefore, it is necessary to establish a more efficient method to achieve higher SSCT without any suffering or mortality of the animals. Methods: A solution of busulfan, ranging from 25 μg/20 μl to 100 μg/20 μl in 50 % DMSO was used for this study. Each testis received two diagonally opposite injections of 10 μl each. Only DMSO was used as control. Germ cell depletion was checked every 15 days. GFP-expressing SSC from transgenic donor mice C57BL/6-Tg (UBC-GFP) 30Scha/J were transplanted into busulfan-treated testis. Two months after SSCT, mice were analyzed for presence of colonies of donor-derived SSC and their ability to generate offspring. Results: A dose of 75 μg of busulfan resulted in reduction of testis size and depletion of the majority of Gc of testis in all mice within 15 days post injection without causing mortality or a cytotoxic effect in other organs. Two months after SSCT, colonies of donor-derived Gc-expressing GFP were observed in recipient testes. When cohabitated with females, donor-derived offspring were obtained. By our method, 71 % of transplanted males sired transgenic progeny as opposed to 5.5 % by previously described procedures. About 56 % of progeny born were transgenic by our method as opposed to 1.2 % by the previously reported methods. Conclusions: We have established an efficient method of generating a germ cell-depleted animal model by using a lower dose of busulfan, injected through two diagonally opposite sites in the testis, which allows efficient colonization of transplanted SSC resulting in a remarkably higher proportion of donor-derived offspring generation

Homeobox transcription factor Meis1 is crucial to Sertoli cell mediated regulation of male fertility

BackgroundInfertility has become a global phenomenon and constantly declining sperm count in males in modern world pose a major threat to procreation of humans. Male fertility is critically dependent on proper functioning of testicular Sertoli cells. Defective Sertoli cell proliferation and/or impaired functional maturation may be one of the underlying causes of idiopathic male infertility. Using high-throughput "omics" approach, we found binding sites for homeobox transcription factor MEIS1 on the promoters of several genes up-regulated in pubertal (mature) Sertoli cells, indicating that MEIS1 may be crucial for Sertoli cell-mediated regulation of spermatogenesis at and after puberty.ObjectiveTo decipher the role of transcription factor MEIS1 in Sertoli cell maturation and spermatogenesis.Materials and methodsSc-specific Meis1 knockdown (KD) transgenic mice were generated using pronuclear microinjection. Morphometric and histological analysis of the testes from transgenic mice was performed to identify defects in spermatogenesis. Epididymal sperm count and litter size were analyzed to determine the effect of Meis1 knockdown on fertility.ResultsSertoli cell (Sc)-specific Meis1 KD led to massive germ cell loss due to apoptosis and impaired spermatogenesis. Unlike normal pubertal Sc, the levels of SOX9 in pubertal Sc of Meis1 KD were significantly high, like immature Sc. A significant reduction in epididymal sperm count was observed in these mice. The mice were found to be infertile or sub-fertile (with reduced litter size), depending on the extent of Meis1 inhibition.DiscussionThe results of this study demonstrated for the first time, a role of Meis1 in Sc maturation and normal spermatogenic progression. Inhibition of Meis1 in Sc was associated with deregulated spermatogenesis and a consequent decline in fertility of the transgenic mice.ConclusionsOur results provided substantial evidence that suboptimal Meis1 expression in Sc may be one of the underlying causes of idiopathic infertility

Reduction in <i>DKK3</i> causes disruption of seminiferous tubules.

<p>(A) Seminiferous tubules of ten weeks old DKDM showing sloughing of Gc (shown by arrow), giant vacuoles (shown by arrowhead) and tubular degeneration. In some of the tubules, sperm were also present. However, the age matched scrambled <i>DKK3</i> mice showed normal spermatogenesis. Scale bar: 50 µm. (B) Seminiferous tubular diameter (in µm) of control mice generated using scrambled <i>DKK3 shRNA</i> construct (open bar) and DKDM (hatched bar) at ten weeks of age. Round and oval tubules were considered for plotting data from three individual mice of each group under equal area of observation (per field) as seen under magnification 20×. Data is represented as mean +/− SEM in each bar. (*p<0.05). (C) Number of normal tubules (dotted bar) and degenerated tubules (crossed bar) in control mice generated using scrambled <i>DKK3 shRNA</i> construct and DKDM at ten weeks of age. Data is plotted by counting normal and degenerated tubules of three individual mice of each group under equal area of observation (per field) as seen under magnification 20×. Data is represented as mean +/− SEM in each bar. (*p<0.05). (D) Seminiferous tubules of ten weeks old control mice generated using scrambled <i>DKK3 shRNA</i> construct and DKDM supplemented with T. T replaced DKDM did not show restoration of normal spermatogenesis in seminiferous tubules. Sloughing of Gc (shown by arrow), giant vacuoles (shown by arrowhead) and tubular degeneration was still seen, Scale bar: 50 µm. All these images are representatives of atleast three random visual fields obtained from atleast three or more animals of each group (Scrambled, DKDM and T replaced DKDM). (E) Quantification of apoptotic Gc in the seminiferous tubules of WT mice and DKDM at ten weeks of age. Data is plotted from random visual fields obtained from atleast three or more individual mice of each group under equal area of observation (per field) as seen under magnification 20×. Data is represented as mean +/− SEM in each bar. (*p<0.05).</p

A model of DKK3 mediated regulation of Sc maturation and spermatogenesis.

<p>In DKDM, LRP6 becomes available because of diminished <i>DKK3</i> levels. Available <i>LRP6</i> binds to Frizzled receptor forming <i>Fz-</i>LRP6 complex augmenting WNT signaling. As a result, <i>GSK-3β</i> is recruited to Frizzled receptor reducing its availability for binding to β-CATENIN, thereby minimizing phosphorylation and degradation of β-CATENIN. Accumulated cytoplasmic β-CATENIN translocates to nucleus and augments expression of different genes, including that of itself, WNT4 and MIS in Sc. Elevated levels of MIS interferes with the Sc maturation, hence, disturbing the balance between spermatogonial proliferation and differentiation leading to subfertility and/or infertility. Additionally, <i>MIS</i> is known to inhibit T production via suppression of CYP17 activity in Lc which might be the reason for reduced T levels in such mice. (eS-elongated spermatids, SSC-spermatogonial stem cells, Fz-Frizzled, rS-round spermatid, SpC-spermatocytes, PTc-peritubular cell, Tcf/Lcf -transcription factors).</p

Characterization of DKDM.

<p>(A) PCR results using gDNA obtained from tail biopsies of progeny generated by crossing electroporated male and WT female mice. Lane 1: 100 bp marker, Lane 2: gDNA of WT mice, Lane 3–12: gDNA of F1 progeny, Lane 13: <i>DKK3</i> shRNA construct. (B) Slot Blot analysis of PCR positive samples of F1 progeny. DNA samples of PCR positive and WT mice (negative control) were hybridized with transgene specific probe. <i>DKK3</i> shRNA construct was used as a positive control. Sample 1 - <i>DKK3</i> shRNA construct, samples 2–14 - gDNA from PCR positive mice, samples 15 and 16 - gDNA from two different WT mice. (C) Real time PCR of <i>DKK3</i> mRNA in the testes of WT mice (open bar), scrambled <i>DKK3</i> (dotted bar) and DKDM (hatched bar) at ten weeks of age. Data are represented as mean +/− SEM (*p<0.05, n = 3). (D) Relative quantity of <i>DKK3</i> mRNA expression in Sc from WT mice (open bar) and DKDM (hatched bar). Real time PCR data of Sc isolated from four or more individual mice of each group is represented as mean +/− SEM (*p<0.05). (E) Western blot analysis of DKK3 from the testes of WT mice and DKDM at ten weeks of age. WT1 and WT2 represents testicular lysates from two different wild type mice, D1–D3 represents testicular lysates from three individual DKDM. The quantity of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as a housekeeping gene is shown in the lower panel. Note: equal amount of protein was loaded in each well. (F) Immunohistochemical localization of DKK3 in testicular sections showing fluorescence and merged images of WT mice (showing higher expression of DKK3) and F1 generation of DKDM (showing diminished expression of DKK3) at ten weeks of age, Scale bar: 50 µm. Inset in the merged image of WT mice shows magnified area of the boxed region. All these images are representatives of atleast three random visual fields obtained from atleast three or more animals of each group (WT and DKDM).</p

<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

DKK3 mediated regulation of β-CATENIN activity in Sc.

<p>(A) Relative quantity of <i>β-CATENIN</i> mRNA levels expressed in cultured Sc isolated from WT mice (open bar) and DKDM (hatched bar). Real time PCR data of Sc isolated from four or more individual mice of each group is represented as mean +/− SEM (*p<0.05). (B) Nuclear localization of β-CATENIN in the purified cultures of Sc isolated from WT mice and DKDM. Nuclear localization of β-CATENIN in Sc of DKDM can be seen. Merged image represents nuclear localization of β-CATENIN (green) along with nuclear staining with DAPI (blue). All these images are representatives of atleast three random visual fields obtained from the Sc isolated from four or more individual mice of each group. Scale bar: 50 µm. (C) Western blot analysis of MIS from the testes of WT mice and DKDM at ten weeks of age. Lanes 1–4 represents testicular lysates from four different WT mice, Lanes 5–8 represents testicular lysates from four different DKDM. The quantity of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as a housekeeping gene is shown in the lower panel. Note: equal amount of protein was loaded in each well.</p

Regulation of WNT signaling by <i>DKK3</i>.

<p>(A) Relative quantity of <i>WNT-4</i> mRNA levels expressed in the testes of WT mice (open bar) and individual DKDM (hatched bar) at ten weeks of age. Real time PCR data from the testicular samples of three animals are represented as mean +/− SEM in each bar. (*p<0.05). (B) Relative quantity of <i>β-CATENIN</i> mRNA levels expressed in the testes of WT mice (open bar) and DKDM (hatched bar) at ten weeks of age. Real time PCR data from the testicular samples of three animals are represented as mean +/− SEM in each bar (*p<0.05). (C) Immunohistochemical localization of β-CATENIN in the testicular sections of WT mice and DKDM showing various florescent and merged images. Nuclear localization of β-CATENIN in Sc of DKDM can be seen. Inset in the merged images of DKDM shows magnified area of the boxed regions. All these images are representatives of atleast three random visual fields obtained from atleast three or more animals of each group (WT and DKDM). Left panel florescent and merged images of WT and DKDM, Scale bar: 50 µm. Right panel florescent and merged images of WT and DKDM, Scale bar: 20 µm.</p