Total Germ Cell Research Articles

Developmental expression patterns of chemokines CXCL11, CXCL12 and their receptor CXCR7 in testes of common marmoset and human.

The chemokine receptor CXCR7 interacts with the chemokines CXCL11 and CXCL12. During development, this ligand receptor system (C-X-C) provokes cell-type-specific responses in terms of migration, adhesion or ligand sequestration. It is active in zebrafish and rodents but no data are available for its presence or function in primate testes. Real-time quantitative polymerase chain reaction was performed in monkeys to detect CXCL11, CXCL12 and CXCR7. At the protein level, CXCL12 and CXCR7 were localized in the testes of the marmoset (Callitrix jacchus) whereas CXCR7 patterns were determined for various stages in human testes. Morphometry and flow cytometry were applied to quantify CXCR7-positive cells in monkeys. Transcript levels and protein expression of CXCR7 were detectable throughout testicular development. In both species, CXCR7 protein expression was restricted to premeiotic germ cells. In immature marmoset testes, 69.9% ± 9% of the total germ cell population were labelled for CXCR7, whereas in the adult, 4.7% ± 2.7% were positive for CXCR7. CXCL12 mRNA was detectable in all developmental stages in marmosets. The CXCL12 protein was exclusively localized to Sertoli cells. This pattern of CXCL12/CXCR7 indicates their involvement in regulatory processes that possibly orchestrate the interaction between undifferentiated germ cells and Sertoli cells.

What lessons can be learned from testicular histology in undescended testes?

Over the last 40-50 years studies involving thousands of testicular biopsies in boys with cryptorchidism have contributed to our knowledge of testicular histopathology and our understanding of the effects of cryptorchidism on the normal development of the germinal epithelium. Growth and maturation of germ cells and Leydig cells are crucial to allow boys to reach normal fertility potential. The following aberrations in testicular development are seen in cryptorchid testes: a decrease in total germ cell numbers, failure of fetal gonocytes (stem cells) to transform into adult dark (Ad) spermatogonia, failure for Ad spermatogonia to mature into primary spermatocytes, Leydig cell hypoplasia, and testicular fibrosis. All of these findings have been found to have a strong negative correlation with a boy’s age at the time of orchidopexy. Some of these findings have prognostic significance in regards to fertility potential especially when coupled with key clinical findings such as hormonal findings, age at orchidopexy, testicle size, laterality and location of cryptorchid testes. This review focuses on key lessons learned from testicular histology in cryptorchid testes.

Pre- and postoperative status of gonadotropins (FSH and LH) and inhibin-B in relation to testicular histopathology at orchiopexy in infant boys with unilateral undescended testes

In recent publications of boys with cryptorchidism, gonadotropins are higher and serum inhibin-B lower than normal. To some extent, serum values of inhibin-B reflect the state of germinative epithelium in cryptorchid testes. The aim of the present study was to evaluate the impact of unilateral orchiopexy on levels of gonadotropins and inhibin-B and correlate the hormone findings to the histopathology of the unilateral undescended testis. 50 boys (mean age: 1 year and 2 months) operated for unilateral cryptorchidism had blood samples for serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and inhibin-B taken preoperatively and 3 months to 2 years postoperatively. Testicular biopsies were performed at orchiopexy. The total germ cell number per transverse tubule and presence of adult dark spermatogonia were estimated. Preoperatively, 8 patients had impaired inhibin-B levels. In contrast with the expected normal physiological decline, 16 patients had an absolute rise in serum inhibin-B level postoperatively. The 4 patients who normalized the preoperative impaired serum level of inhibin-B all had low germ cell number in the testis at time of operation. Nine patients had preoperatively elevated serum levels of both LH 0.5-2.3 IU/l (mean: 1.0 IU/l) and FSH 1.3-2.2 IU/l (mean: 1.6 IU/l). Eight of these patients normalized both LH and FSH serum values postoperatively. None of the 4 boys with impaired inhibin-B level that did not normalize after surgery had elevated gonadotropins. Orchiopexy in unilateral cryptorchidism had a clear impact on the serum level of inhibin-B and gonadotropins of 32% and 16%, respectively.

Perinatal germ cell development and differentiation in the male marmoset (Callithrix jacchus): similarities with the human and differences from the rat

STUDY QUESTIONIs perinatal germ cell (GC) differentiation in the marmoset similar to that in the human?SUMMARY ANSWERIn a process comparable with the human, marmoset GC differentiate rapidly after birth, losing OCT4 expression after 5–7 weeks of age during mini-puberty.WHAT IS KNOWN ALREADYMost of our understanding about perinatal GC development derives from rodents, in which all gonocytes (undifferentiated GC) co-ordinately lose expression of the pluripotency factor OCT4 and stop proliferating in late gestation. Then after birth these differentiated GC migrate to the basal lamina and resume proliferation prior to the onset of spermatogenesis. In humans, fetal GC differentiation occurs gradually and asynchronously and OCT4+ GC persist into perinatal life. Failure to switch off OCT4 in GC perinatally can lead to development of carcinoma in situ (CIS), the precursor of testicular germ cell cancer (TGCC), for which there is no animal model. Marmosets show similarities to the human, but systematic evaluation of perinatal GC development in this species is lacking. Similarity, especially for loss of OCT4 expression, would support use of the marmoset as a model for the human and for studying CIS origins.STUDY DESIGN, SIZE AND DURATIONTestis tissues were obtained from marmosets (n = 4–10 per age) at 12–17 weeks' gestation and post-natal weeks 0.5, 2.5, 5–7, 14 and 22 weeks, humans at 15–18 weeks' gestation (n = 5) and 4–5 weeks of age (n = 4) and rats at embryonic day 21.5 (e21.5) (n = 3) and post-natal days 4, 6 and 8 (n = 4 each).PARTICIPANTS/MATERIALS, SETTING AND METHODSTestis sections from fetal and post-natal marmosets, humans and rats were collected and immunostained for OCT4 and VASA to identify undifferentiated and differentiated GC, respectively, and for Ki67, to identify proliferating GC. Stereological quantification of GC numbers, differentiation (% OCT4+ GC) and proliferation were performed in perinatal marmosets and humans. Quantification of GC position within seminiferous cords was performed in marmosets, humans and rats.MAIN RESULTS AND ROLE OF CHANCEThe total GC number increased 17-fold from birth to 22 post-natal weeks in marmosets; OCT4+ and VASA+ GC proliferated equally in late gestation and early post-natal life. The percentage of OCT4+ GC fell from 54% in late fetal life to <0.5% at 2.5 weeks of age and none were detected after 5–7 weeks in marmosets. In humans, the percentage of OCT4+ GC also declined markedly during the equivalent period. In marmosets, GC had begun migrating to the base of seminiferous cords at ∼22 weeks of age, after the loss of GC OCT4 expression.LIMITATIONS, REASONS FOR CAUTIONThere is considerable individual variation between marmosets. Although GC development in marmosets and humans was similar, there are differences with respect to proliferation during fetal life. The number of human samples was limited.WIDER IMPLICATIONS OF THE FINDINGSThe similarities in testicular GC differentiation between marmosets and humans during the perinatal period, and their differences from rodents, suggest that the marmoset may be a useful model for studying the origins of CIS, with relevance for the study of TGCC.STUDY FUNDING/COMPETING INTERESTSThis work was supported by Grant G33253 from the Medical Research Council, UK. No external funding was sought and there are no competing interests.

Undescended Testis Histology Correlation with Adult Hormone Levels and Semen Analysis

Cryptorchidism has been associated with infertility. We hypothesize that a positive correlation exists between testicular histopathology at orchiopexy and future fertility potential in patients with cryptorchidism. Patients with cryptorchidism who underwent orchiopexy with bilateral testis biopsies were followed into adulthood. Testis histology was stratified into groups based on total germ cells per tubule and adult dark spermatogonia per tubule. After age 18 years, patients underwent hormonal testing and semen analysis. Mean semen analysis parameters and hormone levels were compared among histopathology groups. A total of 91 patients with unilateral undescended testes and 19 with bilateral undescended testes had data for review. No significant differences in semen analysis parameters were seen among the germ cells per tubule groups. In unilateral undescended testis, sperm density and sperm count in the abnormal adult dark spermatogonia per tubule group remained within normal range but were significantly decreased (p = 0.005 and p = 0.028). Follicle-stimulating hormone levels were significantly higher in patients with unilateral undescended testis with abnormal adult dark spermatogonia per tubule but remained within normal range (p = 0.009). Sperm density was below normal range and was significantly decreased in the abnormal adult dark spermatogonia per tubule group in the bilateral undescended testes cohort (p = 0.0496). In bilateral undescended testes follicle-stimulating hormone level, sperm count and percent motility in the abnormal adult dark spermatogonia per tubule group were outside normal clinical range but these results were not statistically significant (p = 0.07-0.2). Total germ cell histopathology at the time of orchiopexy was not associated with significant changes in hormone levels or semen analysis results in adulthood. Testis biopsy at orchiopexy may be limited in predicting future fertility in unilateral undescended testis but more clinically useful in predicting fertility potential for those with bilateral undescended testes.

Apoptosis-mediated seasonal testicular regression in the Japanese Jungle crow (Corvus macrorhynchos)

The present study investigated effects of apoptosis observed during seasonal testicular regression in Japanese Jungle Crows. The study was conducted during January to June 2008, 2009. Testes from adults captured during non-breeding (January), prebreeding (February to mid-March), main-breeding (late March to early May), transition (mid-May to late May), and post-breeding (June) seasons were analyzed. Apoptosis was assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Paired-testis volume increased 95-fold from the non-breeding to the main-breeding season (P < 0.05), and subsequently decreased 26-fold from the main breeding to the post-breeding season (P < 0.05). Testicular activity was evaluated from the total germ cell count and sperm index, which increased 42- and 5-fold, respectively, in the main-breeding season, and subsequently decreased 33- and 5-fold in the post-breeding season. In testes, TUNEL-positive germ cells were at low levels in the non-breeding season, absent in the prebreeding and the main-breeding seasons, and highest in mid-May (P < 0.05). In contrast, TUNEL-positive Sertoli cells occurred only in late-April. In addition, TUNEL-positive fibroblast-like cells were observed in the outer zone of the tunica albuginea in the post-breeding season. Collectively, these data suggested that the seasonal rise in the testicular competence occurred slowly in Japanese Jungle Crows; however, testis function was terminated rapidly after the breeding season. Furthermore, we concluded, similar to other avian species, Sertoli cell apoptosis followed by massive germ cell death was responsible for rapid testicular regression in Jungle Crows.

Histological Examination of Solitary Contralateral Descended Testis in Congenital Absence of Testis

Congenital absence of the testis is believed to be secondary to prenatal torsion, differing from the isolated undescended testis. We determined whether congenital absence of the testis is associated with abnormal histology in the solitary contralateral descended testis. A total of 239 boys with a primary diagnosis of unilateral absent testis underwent orchiectomy and testis biopsy. Germ cell counts were compared between solitary contralateral descended testes and contralateral descended testes in a randomly selected, age matched cohort of patients with unilateral undescended testes. Subanalyses evaluating hypertrophic testes and hypertrophic prepubertal testes between the study groups were performed. The solitary contralateral descended testis group exhibited a significantly greater volume (p <0.001) and a significantly greater germ cell count (p = 0.001). In the hypertrophied testes there was a greater gonocyte count (p = 0.02), greater percentage of gonocytes (p = 0.02), greater primary spermatocyte count (p = 0.04) and greater percentage of primary spermatocytes (p = 0.03). No significant differences in adult dark spermatogonia or Leydig cells were detected. Primary spermatocytes did not differ significantly in prepubertal patients. The solitary contralateral descended testis exhibits increased volume, increased germ cell proliferation and dissimilar maturation patterns compared to the contralateral descended testis in unilateral cryptorchidism. These findings support prenatal torsion rather than endocrinopathy as the etiology for the congenitally absent testis. In the postpubertal solitary contralateral descended testis more germ cell maturation is seen and primary spermatocytes account for the increased total germ cell count. Patients with a solitary testis are likely not at additional risk for infertility.

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.

Histological Findings in Patients With Cryptorchidism and Testis-Epididymis Nonfusion

Fusion anomalies of the testis and epididymis are associated with cryptorchidism. The bilateral histology of the cryptorchid testis associated with the nonfused epididymis has not been reported previously. We retrospectively reviewed patients who presented with unilateral undescended testes and underwent bilateral testis biopsy at orchiopexy between 1982 and 2008. Testes were stratified into groups based on degree of testis-epididymis nonfusion. Age at surgery, testicular volume, testicular position, total germ cells per tubule and adult dark spermatogonia per tubule were compared among all groups. A total of 2,660 testes were eligible for review, of which 2,425 had normal fusion (group 1), 55 had epididymal head nonfusion (group 2), 119 had epididymal tail nonfusion (group 3) and 61 had complete nonfusion (group 4). With increasing degrees of nonfusion trends toward younger age, smaller testicular volume and higher preoperative position were observed. However, testis-epididymis nonfusion was not a significant predictor of abnormal germ cells per tubule or adult dark spermatogonia per tubule in undescended testes and contralateral descended testes. Fusion anomalies are associated with smaller, higher testes with no significant abnormalities in germ cells per tubule or adult dark spermatogonia per tubule. Testis-epididymis nonfusion is not a reliable predictor of reduced histological findings, and should not be a strong consideration when counseling patients and their families about future fertility, especially in instances of complete nonfusion.

Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro

Spermatogonial stem cells (SSCs) support life-long spermatogenesis by self-renewing and producing spermatogonia committed to differentiation. In vitro, SSCs form three-dimensional spermatogonial aggregates (clusters) when cultured with glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2); serial passaging of clusters results in long-term SSC maintenance and expansion. However, the role of these growth factors in controlling patterns of SSC division and fate decision has not been understood thoroughly. We report here that in a short-term culture, GDNF and FGF2 increase the number of dividing SSCs, but not the total SSC number, compared to a no-growth-factor condition. Since the total germ cell number increases with growth factors, these results suggest that GDNF and FGF2 promote a SSC division pattern that sustains the size of the stem cell pool while generating committed progenitors. Our data also show that SSC numbers increase when the cluster structure is disintegrated and cell–cell interaction in clusters is disrupted. Collectively, these results suggest that in this culture system, GDNF and FGF2 stimulate SSC divisions that promote self-renewal and differentiation in the SSC population, and imply that the destruction of the cluster structure, a potential in vitro niche, may contribute to SSC expansion.

Retinoblastoma 1 Protein Modulates XY Germ Cell Entry into G1/G0 Arrest During Fetal Development in Mice1

During mouse germ cell development, the first sign of sex differentiation occurs when XY germ cells enter G(1)/G(0) arrest from 12.5 days postcoitum (dpc). Retinoblastoma 1 (RB1), a potent cell cycle regulator, was investigated in XY germ cell arrest by studying germ cell proliferation in Rb1(-/-) mutant mouse embryos. Because mice homozygous for the Rb1 deletion die in utero around 14.5 dpc, we used ex vivo culture techniques to allow analysis of developing gonads to 16.5 dpc. In Rb1(-/-) gonads, we observed normal somatic cell development, assessed by immunofluorescence for markers HSD3B1 and anti-Müllerian hormone. However, at 14.5 dpc, when wild-type XY germ cells had arrested, we could detect actively proliferating germ cells using the proliferation markers MKI67, pHH3, and bromodeoxyuridine incorporation. The increased proliferation was reflected with a trend of increased germ cell number and expression of germ cell markers Ddx4 and Pou5f1 in the Rb1(-/-) testes. By 16.5 dpc, this phenotype was resolved such that the entire germ cell population had entered G(1)/G(0) arrest, although the total germ cell number remained elevated. At each stage analyzed, we saw no increase in expression of RB1 family members Rbl1 and Rbl2 in the Rb1(-/-) testes, but we saw a significant increase of cyclin-dependent kinase (CDK) inhibitor Cdkn1b and Cdkn2b expression. We conclude that Rb1 is required for correct germ cell entry into G(1)/G(0) arrest in the wild-type gonad at 14.5 dpc, but in its absence, upregulation of other cell cycle suppressors, including Cdkn1b and Cdkn2b, can induce delayed germ cell arrest.

Calculation of spermatogenic transformations based on dual-flow cytometric analysis of testicular tissue in seasonal breeders.

The percentages of somatic and spermatogenic cells were quantified at different time intervals in testes of three seasonal breeders: roe deer, brown hare and mink. The haploid, diploid and tetraploid cells were monitored by one-parameter flow cytometry. Somatic and spermatogenic cells were distinguished by dual-parameter flow cytometry after DNA staining and selective labelling of the somatic cells with an antivimentin antibody. The portions of spermatogenic diploid and tetraploid cells were used to calculate total germ cell transformation (haploid:diploid cell ratio) as well as meiotic transformation (haploid:tetraploid cell ratio). During the breeding season the means of both ratios were 10.27 and 38.42 in roe deer, 7.55 and 15.81 in brown hare, and 11.25 and 53.47 in mink. The equivalent quotients calculated on the base of total diploid and tetraploid cells were considerably lower: 4.41 and 7.79, 5.13 and 7.86, 5.68 and 13.75, respectively. Portions as well as proportions of germinative and somatic cells changed during the annual cycle. The results demonstrate that the distinction of somatic and germinative cells in flow cytometric studies of spermatogenesis is a prerequisite for exact calculations of mitotic and meiotic processes and their alterations under different conditions.

Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal

Self-renewal and differentiation of spermatogonial stem cells (SSCs) provide the foundation for testis homeostasis, yet mechanisms that control their functions in mammals are poorly defined. We used microarray transcript profiling to identify specific genes whose expressions are augmented in the SSC-enriched Thy1(+) germ cell fraction of mouse pup testes. Comparisons of gene expression in the Thy1(+) germ cell fraction with the Thy1-depleted testis cell population identified 202 genes that are expressed 10-fold or higher in Thy1(+) cells. This database provided a mining tool to investigate specific characteristics of SSCs and identify novel mechanisms that potentially influence their functions. These analyses revealed that colony stimulating factor 1 receptor (Csf1r) gene expression is enriched in Thy1(+) germ cells. Addition of recombinant colony stimulating factor 1 (Csf1), the specific ligand for Csf1r, to culture media significantly enhanced the self-renewal of SSCs in heterogeneous Thy1(+) spermatogonial cultures over a 63-day period without affecting total germ cell expansion. In vivo, expression of Csf1 in both pre-pubertal and adult testes was localized to clusters of Leydig cells and select peritubular myoid cells. Collectively, these results identify Csf1 as an extrinsic stimulator of SSC self-renewal and implicate Leydig and myoid cells as contributors of the testicular stem cell niche in mammals.

Cryptorchid testis histopathology in myelomeningocele patients

Purpose Cryptorchidism occurs in 25% of boys with myelomeningocele (MMC) compared to 3% of the general population. Testicular biopsy histopathology correlates with future sperm counts. We studied testicular histology in boys with cryptorchidism and MMC to investigate if the MMC influences histological findings. Materials and methods The study group consisted of six patients with MMC and undescended testis (UDT) who underwent orchiopexy and bilateral testis biopsy. Twelve testicular biopsies from six patients were compared to 40 biopsies from 20 UDT-only controls. Total germ cell count per tubule (TGC/T) and the percentage of adult dark spermatogonia (%Ad) in undescended and contralateral descended testes from the patients were compared with controls. Results In the study group, two had total absence of germ cells (TGC/T = 0) and three had severely reduced germ cells (TGC/T < 0.2). Four had total absence of Ad spermatogonia and the remaining two had severely reduced Ad spermatogonia (%Ad = 5). The mean TGC/T and %Ad in patients with UDT and MMC were conspicuously lower than controls. The differences did not reach statistical significance ( P = 0.09–0.29). Conclusion These results suggest that patients with both MMC and UDT have a more severe reduction in total number and more severely delayed maturation of germ cells than do patients with UDT alone. With only six patients in this study, there was not the power to detect statistical significance. In addition to the reproductive problems due to erection and ejaculatory dysfunction in patients with MMC, this severe testicular histopathology may increase the risk of subfertility.

Spermatogenesis and Sertoli Cell Activity in Mice Lacking Sertoli Cell Receptors for Follicle-Stimulating Hormone and Androgen

Spermatogenesis in the adult male depends on the action of FSH and androgen. Ablation of either hormone has deleterious effects on Sertoli cell function and the progression of germ cells through spermatogenesis. In this study we generated mice lacking both FSH receptors (FSHRKO) and androgen receptors on the Sertoli cell (SCARKO) to examine how FSH and androgen combine to regulate Sertoli cell function and spermatogenesis. Sertoli cell number in FSHRKO-SCARKO mice was reduced by about 50% but was not significantly different from FSHRKO mice. In contrast, total germ cell number in FSHRKO-SCARKO mice was reduced to 2% of control mice (and 20% of SCARKO mice) due to a failure to progress beyond early meiosis. Measurement of Sertoli cell-specific transcript levels showed that about a third were independent of hormonal action on the Sertoli cell, whereas others were predominantly androgen dependent or showed redundant control by FSH and androgen. Results show that FSH and androgen act through redundant, additive, and synergistic regulation of spermatogenesis and Sertoli cell activity. In addition, the Sertoli cell retains a significant capacity for activity, which is independent of direct hormonal regulation.

Expression of leptin and leptin receptor in the testis of fertile and infertile patients

The aim of our study was to investigate the relationships between the expression of leptin, leptin receptor in the testis and spermatogenesis, and testosterone (T) concentration in infertile men. Testicular tissue samples were collected from the testes of five fertile volunteers, eight patients with obstructive azoospermia (OA), six patients with Sertoli cell-only syndrome (SCO) and 32 oligospermic patients with varicocele testis. In testicular tissue, leptin and leptin receptor were identified by staining with polyclonal antibodies. Serum follicle stimulating hormone, lutenising hormone (LH), and T were determined by chemiluminescence assays. Leptin was expressed on germ cells, mainly on spermatocytes. The ratio of immunostained germ cells to total germ cells was inversely correlated with the concentration of T (r = -0.32, P = 0.01), sperm concentration (r = -0.51, P = 0.002) and Johnsen's score (r = -0.44,P = 0.005). In contrast, leptin receptor immunostained cells were found in the interstitium, primarily in Leydig cells. Leptin receptor expression on Leydig cells was inversely correlated with serum T concentration (r = -0.50, P < 0.001). The dysfunction of spermatogenesis is associated with an increase in leptin and leptin receptor expression in the testis.

NF-kappaB Activation Elicited by Ionizing Radiation Is Proapoptotic in Testis1

The transcription factor NF-kappaB modulates apoptotic machinery following activation by the IkappaB kinase (IKK) complex. Inhibiting activity of one of the catalytic subunits of the IKK complex, IKKbeta (also known as IKBKB and IKK2) severely inhibits NF-kappaB nuclear translocation in response to most stimuli, including ionizing radiation. Doubly floxed Ikkbeta(F/F) mice (control) were compared to haplo-insufficient Ikkbeta(F/)(delta) mice (NF-kappaB knockdown) to examine the in vivo apoptotic role of NF-kappaB in the testis. Although Ikkbeta(F/F) control adult mice had spermatid head counts and testis and body weights similar to Ikkbeta(F/)(delta) mice, cellular stress in the form of ionizing radiation elicited a differential phenotype. Lower body exposure to 5 Gy of ionizing radiation induced a greater NF-kappaB activation in Ikkbeta(F/F) than in Ikkbeta(F/)(delta) mice. In addition, exposure to ionizing radiation resulted in fewer apoptotic germ cells 3, 6, and 12 h after injury in NF-kappaB knockdown mice than in controls, concomitant with the reduced cleavage of caspases 3 and 9 at 3 h. There was also a reduction in total germ cells lost after radiation with NF-kappaB inhibition. Correspondingly, real-time RT-PCR showed a significant reduction in Cdnk1a (also known as p21) and Fasl expression 3 and 6 h, respectively, after irradiation in Ikkbeta(F/)(delta) compared to control testes. These data indicate that, despite acting in an antiapoptotic manner in many tissue types, NF-kappaB is proapoptotic in modulating the germ cell response to ionizing radiation.

Oligozoospermia with normal fertility in male mice lacking the androgen receptor in testis peritubular myoid cells

Androgens and the androgen receptor (AR) play important roles in the testes. Previously we have shown that male total AR knockout (T-AR-/y) mice revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in particular types of testicular cells remain unclear. Using a Cre-loxP conditional knockout strategy, we generated a tissue-selective knockout mouse with the AR gene deleted in testis peritubular myoid cells (PM-AR-/y). Phenotype analyses showed that PM-AR-/y mice were indistinguishable from WT AR (AR+/y) mice with the exception of smaller testes size. PM-AR-/y mice have serum testosterone concentrations comparable with AR+/y mice. PM-AR-/y mice have oligozoospermia in the epididymis; however, fertility was normal. Although normal germ cell distribution ratio was found, total germ cell number decreased in PM-AR-/y mice. Further mechanistic studies demonstrated that PM-AR-/y mice have defects in the expression of Sertoli cells' functional marker genes such as tranferrin, epidermal fatty acid-binding protein, androgen-binding protein, and other junction genes including occludin, testin, nectin, zyxin, vinculin, laminingamma3, gelsolin, connection43, and N-cadherin. Furthermore, there were defects in peritubular myoid cell contractility-related genes such as endothelin-1, endothelin receptor A and B, adrenomedullin, adrenomedullin receptor, and vasopressin receptor 1a. Together, our PM-AR-/y mice provide in vivo evidence for the requirement of functional AR in peritubular myoid cells to maintain normal Sertoli cells function and peritubular myoid cell contractility, thus ensuring normal spermatogenesis and sperm output.

Laminin α 3 Forms a Complex with β3 and γ3 Chains That Serves as the Ligand for α 6β1-Integrin at the Apical Ectoplasmic Specialization in Adult Rat Testes

Apical ectoplasmic specialization (ES) is a testis-specific hybrid cell/cell actin-based adherens junction and cell/matrix focal contact anchoring junction type restricted to the interface between Sertoli cells and developing spermatids. Recent studies have shown that laminin gamma3, restricted to elongating spermatids, is a putative binding partner of alpha 6beta 1-integrin localized in Sertoli cells at the apical ES. However, the identity of the alpha and beta chains, which constitute a functional laminin ligand with the gamma3 chain at the apical ES, is not known. Using reverse transcription-PCR and immunoblotting to survey all laminin chains in cells of the seminiferous epithelium, it was noted that alpha 2, alpha 3, beta1, beta2, beta3, and gamma3 chains were found in germ cells, whereas alpha 1, alpha 2, alpha 4, alpha 5, beta1, beta2, gamma1, gamma2, and gamma3 chains were found in Sertoli cells, implying that alpha 3 and beta3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of gamma3. To verify this postulate, recombinant proteins based on domain G of alpha 3 and domain I of beta3 and gamma3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin alpha 3, beta3, and gamma3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with beta1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin alpha 3, beta3, and gamma3, as well as beta1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin alpha 3 or gamma3 IgG, this treatment perturbed adhesion between Sertoli and germ cells (mostly spermatids), leading to germ cell loss from the epithelium. More important, a transient disruption of the blood-testis barrier was also detected.

Reproduction of wild Mongolian gerbils bred in the laboratory with respect to generation and season 2. Spermatogenic activity and testicular testosterone concentration

AbstractTesticular activity of wild and laboratory strains of Meriones unguiculatus and its seasonal dependence was studied. Comparative intraspecific measurements were performed in adult males of the laboratory strain (LAB) and in male offspring of wild Mongolian gerbils (WF-1 to WF-3). All animals were reared under identical conditions including a constant light regime. LAB and WILD were examined in January, July and October. Testicular spermatozoa were counted, proportions of different cell types were analysed using DNA flow cytometry, and total germ cell transformation was calculated from DNA histograms. Intratesticular testosterone concentrations were measured with an enzyme immunoassay. The results showed that differences of testicular activity are dependent on the season as well as the strain and the generation. The season affected testicular parameters in LAB resulting in lowered testosterone and sperm production in July and October, respectively. In contrast, all testicular cell parameters were significantly lower for WF-1 in January compared with October. These seasonal differences were neutralized or diminished in WF-2 and WF-3. In the WILD, total testicular activity was lower and varied more in all months studied, but the differences compared with the values in LAB decreased in the successive generations. WF-3 showed the lowest discrepancy of testis weight and spermatogenic output compared with LAB. The data suggest endogenous cues for seasonal regulation of spermatogenesis, but this endogenous rhythm seems to be subsided or lost rapidly. These findings indicate rapid, adaptive changes in the early offspring generations from wild populations during breeding in the laboratory.