Regulation of neonatal Sertoli cell development by thyroid hormone receptor alpha1

Neonatal hypothyroidism increases adult Sertoli cell populations by extending Sertoli cell proliferation. Conversely, hyperthyroidism induces premature cessation of Sertoli cell proliferation and stimulates maturational events like seminiferous tubule canalization. Thyroid hormone receptors alpha1 and beta1, which are commonly referred to as TRalpha1 and TRbeta1, respectively, are expressed in neonatal Sertoli cells. We determined the relative roles of TRalpha1 and TRbeta1 in the thyroid hormone effect on testicular development and Sertoli cell proliferation using Thra knockout (TRalphaKO), Thrb knockout (TRbetaKO), and wild-type (WT) mice. Triiodothyronine (T3) treatment from birth until Postnatal Day 10 reduced Sertoli cell proliferation to minimal levels in WT and TRbetaKO mice versus that in their untreated controls, whereas T3 had a diminished effect on TRalphaKO Sertoli cell proliferation. Seminiferous tubule patency and luminal diameter were increased in T3-treated WT and TRbetaKO testes. In contrast, T3 had no effect on these parameters in TRalphaKO mice. In untreated adult TRalphaKO mice, Sertoli cell number, testis weight, and daily sperm production were increased or trended toward an increase, but the increase in magnitude was smaller than that seen in WT mice following neonatal hypothyroidism. Conversely, in TRbetaKO mice, Sertoli cell number, testis weight, and daily sperm production were similar to those in untreated WT mice. In addition, Sertoli cell number and testis weight in adult WT and TRbetaKO mice showed comparable increases following hypothyroidism. Our results show that TRalphaKO mice have testicular effects similar to those seen in WT mice following neonatal hypothyroidism and that TRbetaKO mice, but not TRalphaKO mice, have normal Sertoli cell responsiveness to T3. Thus, effects of exogenous manipulation of T3 on neonatal Sertoli cell development are predominately mediated through TRalpha1.     

An exploration of the role of Sertoli cells on fetal testis development using cell ablation strategy

Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh‐Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α‐smooth muscle actin‐labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3β‐HSD double‐positive fetal LCs could be observed in Amh‐Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3β‐HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs’ central role in fetal testis development.     

Collagen1alpha1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1alpha1 (Col1alpha1) promoter (Col1alpha1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1alpha1-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4(Co/Co)). Multiple tissue PCR of Col1alpha1-Cre;Smad4(Co/+)mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1alpha1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1alpha1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.     

Thyroid status during skeletal development determines adult bone structure and mineralization

Childhood hypothyroidism delays ossification and bone mineralization, whereas adult thyrotoxicosis causes osteoporosis. To determine how effects of thyroid hormone (T3) during development manifest in adult bone, we characterized TRalpha1(+/m)beta(+/-) mice, which express a mutant T3 receptor (TR) alpha1 with dominant-negative properties due to reduced ligand-binding affinity. Remarkably, adult TRalpha1(+/m)beta(+/-) mice had osteosclerosis with increased bone mineralization even though juveniles had delayed ossification. This phenotype was partially normalized by transient T3 treatment of juveniles and fully reversed in compound TRalpha1(+/m)beta(-/-) mutant mice due to 10-fold elevated hormone levels that allow the mutant TRalpha1 to bind T3. By contrast, deletion of TRbeta in TRalpha1(+/+)beta(-/ -) mice, which causes a 3-fold increase of hormone levels, led to osteoporosis in adults but advanced ossification in juveniles. T3-target gene analysis revealed skeletal hypothyroidism in TRalpha1(m/+)beta(+/-) mice, thyrotoxicosis in TRalpha1(+/+)beta(-/-) mice, and euthyroidism in TRalpha1(+/)beta(-/-) double mutants. Thus, TRalpha1 regulates both skeletal development and adult bone maintenance, with euthyroid status during development being essential to establish normal adult bone structure and mineralization.     

Testicular shape and its relationship to sperm production in mature Holstein bulls

This study was conducted to determine the relationship between testicular shape, scrotal circumference (SC) and sperm production. Twenty-seven mature Holstein bulls were evaluated subjectively and objectively for testicular shape as indicated by testicular length and width, then placed in 1 of 3 groups. Group 1 contained 17 bulls with a normal ovoid testicular shape and a length to width ratio of 1.61:1 +/- 0.01 (SEM). Group 2 was composed of 4 bulls with a long, slender testicular shape and a length to width ratio of 1.95:1 +/- 0.06 (SEM). Group 3 was comprised of 6 bulls with spheroid-shaped testicles and a length to width ratio of 1.3:1 +/- 0.03 (SEM). All the groups were statistically different for length to width ratios (P < 0.05). Length measurements from cranial to caudal pole of the testis proper were also different between groups (P < 0.05). Width or testicular diameter was different between Group 2 and Group 3 at P < 0.05; however, there was no difference between Group 1 and Group 2 or between Group 1 and Group 3. Predicted volumes and weights of testicles were not significantly different between groups. Scrotal circumference measurements were significantly different between groups (P < 0.05). Group 1 had an average SC of 43.07 +/- 0.36 cm (SEM), Group 2 of 39.33 +/- 1.18 cm (SEM) and Group 3 of 46.22 +/- 0.69 cm (SEM). Sperm production for a twice daily, 2-day-per-week collection schedule revealed a statistically significant difference for sperm output. A total of 2742 ejaculates was evaluated. A total of 1818 ejaculates was evaluated in Group 1, 440 ejaculates in Group 2 and 484 ejaculates in Group 3. The mean spermatozoal harvest per day for Group 1 bulls was 13.62 +/- 0.09 x 10(9) (SEM). Group 2 bulls with the longer-shaped testicles produced 14.82 +/- 0.18 x 10(9) (SEM) spermatozoa per day, and Group 3 bulls, with the more rounded testicle shape and the significantly larger SC produced 11.72 +/- 0.64 x 10(9)(SEM) sperm cells per day. All 3 groups were statistically different at the P = 0.05 level. The results suggest that prediction of sperm production may be dependent on factors other than SC, testicular volume, or weight. Testicular shape may influence sperm output in mature Holstein bulls.     

Cardiac glucose utilization in mice with mutated alpha- and beta-thyroid hormone receptors

Abnormal thyroid function is usually associated with altered cardiac function. Mutations in the thyroid hormone (TH)-binding region of the TH beta-receptor (TRbeta) that eliminate its TH-binding ability lead to the thyroid hormone resistance syndrome (RTH) in humans, which is characterized by high blood TH levels, goiter, hyperactivity, and tachycardia. Mice with "knock-in" mutations in the TH alpha-receptor (TRalpha) or TRbeta that remove their TH-binding ability have been developed, and those with the mutated TRbeta (TRbeta(PV/PV)) appear to provide a model for RTH. These two types of mutants show different effects on cerebral energy metabolism, e.g., negligible change in glucose utilization (CMR(Glc)) in TRbeta(PV/PV) mice and markedly reduced CMR(Glc), like that found in cretinous rats, in the mice (TRalpha(PV/+)) with the knock-in mutation of the TRalpha gene. Studies in knockout mice have indicated that the TRalpha may also influence heart rate. Because mutations in both receptor genes appear to affect some parameters of cardiac function and because cardiac functional activity and energy metabolism are linked, we measured heart glucose utilization (HMR(Glc)) in both the TRbeta(PV/PV) and TRalpha(PV/+) mutants. Compared with values in normal wild-type mice, HMR(Glc) was reduced (-77 to -95%) in TRalpha(PV/+) mutants and increased (87 to 340%) in TRbeta(PV/PV) mutants, the degree depending on the region of the heart. Thus the TRalpha(PV/+) and TRbeta(PV/PV) mutations lead, respectively, to opposite effects on energy metabolism in the heart that are consistent with the bradycardia seen in hypothyroidism and the tachycardia associated with hyperthyroidism and RTH.     

Rete testis and the adjacent seminiferous tubules during postembryonic development in mice

Testicular compartment that includes rete testis and the adjacent transitional zone (TZ) of seminiferous tubules has been examined only by light and electron microscopy until now. However, recent data suggest that adult Sertoli cells (SCs) located in this compartment are capable to commence active proliferation both in vitro and in vivo, and hence, are not completely differentiated. The present study is first to investigate mouse rete testis and TZ during the postembryonic development and is intended to determine new protein markers for cells of this compartment, the state of their differentiation, and also their proliferative activity. It was demonstrated that rete testis cells were stained for SC marker Wt1 transiently, until day 25 of postembryonic development, then the staining disappeared. Another SC marker Dmrt1 that involved in the process of SC differentiation was not expressed in the rete testis cells during the postnatal development and in the adult state. One more feature that distinguished rete testis cells from SCs was lower proliferative activity of rete testis cells in 2–6 days old mice. SCs from TZ expressed Wt1 at all ages examined. However, at earlier ages, they were heterogeneous on Dmrt1 expression, and only by day 25, Dmrt1 expression was completely disappeared from TZ SCs. It is interesting that on day 18 when SCs in seminiferous tubules complete differentiation and exit from cell cycle proliferation of TZ SCs was at significantly higher level. It is also showed that in 3D culture, Wt1+ cells isolated from rete testis and TZ of 60 days old GFP male mice were capable to form seminiferous tubules de novo in cooperation with testicular cells from 6 days old mice.     

Deficiency of Macf1 in osterix expressing cells decreases bone formation by Bmp2/Smad/Runx2 pathway

Microtubule actin cross‐linking factor 1 (Macf1) is a spectraplakin family member known to regulate cytoskeletal dynamics, cell migration, neuronal growth and cell signal transduction. We previously demonstrated that knockdown of Macf1 inhibited the differentiation of MC3T3‐E1 cell line. However, whether Macf1 could regulate bone formation in vivo is unclear. To study the function and mechanism of Macf1 in bone formation and osteogenic differentiation, we established osteoblast‐specific Osterix (Osx) promoter‐driven Macf1 conditional knockout mice (Macf1^f/fOsx‐Cre). The Macf1^f/fOsx‐Cre mice displayed delayed ossification and decreased bone mass. Morphological and mechanical studies showed deteriorated trabecular microarchitecture and impaired biomechanical strength of femur in Macf1^f/fOsx‐Cre mice. In addition, the differentiation of primary osteoblasts isolated from calvaria was inhibited in Macf1^f/fOsx‐Cre mice. Deficiency of Macf1 in primary osteoblasts inhibited the expression of osteogenic marker genes (Col1, Runx2 and Alp) and the number of mineralized nodules. Furthermore, deficiency of Macf1 attenuated Bmp2/Smad/Runx2 signalling in primary osteoblasts of Macf1^f/fOsx‐Cre mice. Together, these results indicated that Macf1 plays a significant role in bone formation and osteoblast differentiation by regulating Bmp2/Smad/Runx2 pathway, suggesting that Macf1 might be a therapeutic target for bone disease.     

Disruption of PTH Receptor 1 in T Cells Protects against PTH-Induced Bone Loss

Background

Hyperparathyroidism in humans and continuous parathyroid hormone (cPTH) treatment in mice cause bone loss by regulating the production of RANKL and OPG by stromal cells (SCs) and osteoblasts (OBs). Recently, it has been reported that T cells are required for cPTH to induce bone loss as the binding of the T cell costimulatory molecule CD40L to SC receptor CD40 augments SC sensitivity to cPTH. However it is unknown whether direct PTH stimulation of T cells is required for cPTH to induce bone loss, and whether T cells contribute to the bone catabolic activity of PTH with mechanisms other than induction of CD40 signaling in SCs.

Methodology/Principal Findings

Here we show that silencing of PTH receptor 1 (PPR) in T cells blocks the bone loss and the osteoclastic expansion induced by cPTH, thus demonstrating that PPR signaling in T cells is central for PTH-induced reduction of bone mass. Mechanistic studies revealed that PTH activation of the T cell PPR stimulates T cell production of the osteoclastogenic cytokine tumor necrosis factor α (TNF). Attesting to the relevance of this effect, disruption of T cell TNF production prevents PTH-induced bone loss. We also show that a novel mechanism by which TNF mediates PTH induced osteoclast formation is upregulation of CD40 expression in SCs, which increases their RANKL/OPG production ratio.

Conclusions/Significance

These findings demonstrate that PPR signaling in T cells plays an essential role in PTH induced bone loss by promoting T cell production of TNF. A previously unknown effect of TNF is to increase SC expression of CD40, which in turn increases SC osteoclastogenic activity by upregulating their RANKL/OPG production ratio. PPR-dependent stimulation of TNF production by T cells and the resulting TNF regulation of CD40 signaling in SCs are potential new therapeutic targets for the bone loss of hyperparathyroidism.     

Luteinizing hormone receptor-mediated effects on initiation of spermatogenesis in gonadotropin-deficient (hpg) mice are replicated by testosterone

Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1). whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2). whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1-100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.     

In vitro primary satellite cell growth and differentiation within litters of pigs

Postnatal muscle growth is dependent on satellite cell (SC) proliferation, differentiation and fusion to increase the DNA content of existing muscle fibres and thereby the capacity to synthesize protein. The purpose of the present study was to examine the ability of isolated SCs from low, medium and high weaning weight litter mates of pigs to proliferate and differentiate, and to affect protein synthesis and degradation after fusion into myotubes. At 6 weeks of age, SCs from the lowest weight (LW), medium weight (MW) and highest weight (HW) female pigs within eight litters were isolated. Thereby, eight cultures of SCs were established for each of the three weight groups within litter, representing three groups of SCs from pigs exhibiting differences in postnatal muscle growth performance. Proliferation was estimated as the number of viable cells at different time points after seeding. SC differentiation was evaluated by measuring the activity of the muscle-specific enzyme, creatine phosphokinase, and protein synthesis and degradation were measured by incorporation and release of 3H-tyrosine, respectively. A tendency towards a difference in proliferation between SC cultures was found (P = 0.09). This was evident as the number of viable cells at day 3 was lower in cultures from LW pigs than from HW (P < 0.05) and MW (P < 0.01) pigs. Differentiation was significantly different between cultures (P < 0.05). There was a significant difference between LW and MW cultures at 72 h (P < 0.05), and a tendency towards a difference between LW and HW cultures at 45 h (P = 0.07). Protein synthesis per μg protein or per μg DNA did not differ among SC cultures from LW, MW and HW pigs. Neither did protein degradation rate differ significantly among SC cultures from LW, MW and HW pigs. Overall, the results show that SCs from LW pigs seem to proliferate and differentiate at a slower rate than SCs from MW and HW pigs. The results found in this study show no difference in the ability of SCs to affect protein synthesis or degradation between SCs from litter mates exhibiting different growth rates in vivo.