The predominant testicular gap junctional protein connexin43 (cx43) is located between neighboring Sertoli cells (SCs) and between SCs and germ cells. It is assumed to be involved in testicular development, cell differentiation, initiation, and maintenance of spermatogenesis with alterations of its expression being correlated with various testicular disorders. Because total disruption of the cx43 gene leads to perinatal death, we generated a conditional cx43 knockout (KO) mouse using the Cre/loxP recombination system, which lacks the cx43 gene solely in SCs (SCCx43KO), to evaluate the SC-specific functions of cx43 on spermatogenesis in vivo. Adult SCCx43KO(-/-) mice showed normal testis descent and development of the urogenital tract, but testis size and weight were drastically lower compared with heterozygous and wild-type littermates. Histological analysis and quantitation of mRNA expression of germ cell-specific marker genes revealed a significant reduction in the number of spermatogonia but increased SC numbers/tubule with only a few tubules left showing normal spermatogenesis. Thus, SC-specific deletion of cx43 mostly resulted in an arrest of spermatogenesis at the level of spermatogonia or SC-only syndrome and in intratubular SC clusters. Our data demonstrate for the first time that cx43 expression in SCs is an absolute requirement for normal testicular development and spermatogenesis.

Endometriosis is characterized by presence of endometrial tissue outside the uterus. Prevalence is estimated at 6-10% in the general female population and many patients experience pain and/or infertility. Diagnosis is achieved by laparoscopic intervention followed by histological confirmation of viable endometriotic tissue. Mild cases are managed medically with contraceptive steroids and non-steroidal anti-inflammatory agents. Surgery provides relief to women in pain but symptoms recur in 75% of cases within 2 years. Starting with menstruation, we have categorized endometriosis into six stages, namely (1) shedding of cells, (2) cell survival, (3) escape from immune surveillance, (4) adhesion to peritoneum, (5) angiogenesis and (6) bleeding. In most of these biological processes, which resemble metastasis, transforming growth factor-beta (TGF-betas) and their high-affinity receptors are involved directly or indirectly. TGF-betas are abundantly and differentially expressed in the endometrium under hormonal control. Although they are preferentially synthesized in the stroma, glands and macrophages also secrete TGF-betas into the uterine fluid, where interaction with preimplantation embryos is suspected. Because mRNA and protein expression of all three TGF-betas is increased around menstruation, we suggest that TGF-betas might be involved in initiation of menstruation. Furthermore, because of high postmenstrual TGF-beta3 levels, we suppose that it might participate in scarless postmenstrual regeneration of endometrium. Our suggestions pave the way to novel routes of investigation into the roles of TGF-betas during menstruation and endometriosis.

Macrophages are important in the activation of innate immune responses and in a tissue-specific manner in the maintenance of organ homeostasis. Testicular macrophages (TM), which reside in the testicular interstitial space, comprise the largest leukocyte population in the testes and are assumed to play a relevant function in maintaining testicular immune privilege. Numerous studies have indicated that the interstitial fluid (IF) surrounding the TM has immunosuppressive properties, which may influence the phenotype of TM. However, the identity of the immunosuppressive molecules present in the IF is poorly characterized. We show that the rat testicular IF shifted GM-CSF–induced M1 toward the M2 macrophage phenotype. IF-polarized M2 macrophages mimic the properties of TM, such as increased expression of CD163, high secretion of IL-10, and low secretion of TNF-α. In addition, IF-polarized macrophages display immunoregulatory functions by inducing expansion of immunosuppressive regulatory T cells. We further found that corticosterone was the principal immunosuppressive molecule present in the IF and that the glucocorticoid receptor is needed for induction of the testis-specific phenotype of TM. In addition, TM locally produce small amounts of corticosterone, which suppresses the basal expression of inflammatory genes as a means to render TM refractory to inflammatory stimuli. Taken together, these results suggest that the corticosterone present in the testicular environment shapes the immunosuppressive function and phenotype of TM and that this steroid may play an important role in the establishment and sustenance of the immune privilege of the testis.

BACKGROUND: Human sperm contain similar amounts of protamine-1 (P1) and protamine-2 (P2). Although aberrant protamine ratios have been observed in infertile men, functional evidence is provided by protamine knockout mice exhibiting male infertility. As sperm DNA integrity is known to be linked with DNA fragmentation and apoptosis, we investigated whether the protamine ratio or Bcl2 content represent a reliable biomarker to discriminate fertile and infertile men. METHODS: Real-time quantitative RT-PCR was used for P1, P2 and the apoptotic marker Bcl2 in testicular biopsies (TB; 74 infertile men versus 17 controls) and ejaculates (E; 95 infertile men versus 10 controls). RESULTS: The P1-P2 mRNA ratio differed significantly between groups, namely 1:4 versus 1:3.2 in TB (P 5 0.0038) and 1:1.7 versus 1:1 in E (P 5 0.0002), for infertile men and controls, respectively. Bcl2 mRNA content was correlated with protamine mRNA ratio (P 5 0.0250 for TB; P 5 0.0003 for E). Infertile men exhibit a more than 10-fold (P 5 0.0155 for TB; P 5 7.0 3 10 26 for E) higher Bcl2 mRNA content versus controls. No correlation was found between absolute sperm density and the protamine mRNA ratio or Bcl2 mRNA content. No significant correlation was demonstrated with fertilization rate after ICSI and either protamine ratio or Bcl2 content. CONCLUSIONS: We found significantly aberrant protamine ratios and a higher Bcl2 content in TB and E of infertile men compared to controls, suggesting that these molecules may be useful biomarkers for predicting male infertility.

In the present study the activity of a 20% methanolic extract of stinging nettle roots (Urtica dioica L., Urticaceae) on the proliferative activity of human prostatic epithelial (LNCaP) and stromal (hPCPs) cells was evaluated using a colorimetric assay. A concentration-dependent and significant (p < 0.05) antiproliferative effect of the extract was observed only on LNCaP cells during 7 days, whereas stromal cell growth remained unaltered. The inhibition was time-dependent with the maximum of growth reduction (30%) at a concentration of 1.0E-6 mg/ml on day 5 compared to the untreated control. On day 4 and 6, the reduction in proliferation of LNCaP cells showed the minimal effective dose at 1.0E-9 mg/ml. No cytotoxic effect of ME-20 on cell proliferation was observed. The antiproliferative effect of ME-20 of stinging nettle roots observed both in an in vivo model and in an in vitro system clearly indicates a biologically relevant effect of compounds present in the extract.

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In the present study we examined the expression and release of the extracellular matrix glycoprotein fibronectin (FN) in a prostate cancer cell line (LNCaP) and in primary prostatic stromal cells using the reverse transcription-polymerase chain reaction (RT-PCR) and by an enzyme-linked immunosorbent assay. Perturbation experiments in vitro using antibodies directed against FN and the FN receptor were also performed. Immunohistochemistry was used to show the in vivo distribution of FN and the FN receptor in tissue sections of normal human prostate, benign prostatic hyperplasia, and prostate carcinoma. The expression of the oncofetal FN ED-B segment in benign prostatic hyperplasia and prostate carcinoma tissue was investigated by RT-PCR. The FN mRNA was expressed by LNCaP and primary prostatic stromal cells, respectively. Both cell types released FN into the medium in a time-dependent manner, whereby FN secretion was about 2.5-fold higher in cultures of stromal cells relative to LNCaP cells. Blocking FN with anti-FN antibodies resulted in a significant decrease in cell adhesion for LNCaP cells and a change in morphology for the primary stromal cells. FN was located mainly in the stromal compartment of the prostate, showing a distinct distribution pattern in prostate carcinoma, whereas the FN receptor was detectable only in the prostate epithelia. RT-PCR experiments showed the expression of the oncofetal FN ED-B segment in benign prostatic hyperplasia and prostate carcinoma tissue, with a 3.5-fold higher expression in the prostate carcinoma probes. Our data point to an important role for FN in cell adhesion of prostatic cells and show that an alternatively spliced FN mRNA is upregulated in the pathologically altered human prostate.