Lack of effect on rat testicular organogenesis after in utero exposure to 3-monochloropropane-1,2-diol (3-MCPD).

3-Monochloropropane-1,2-diol (3-MCPD) is a food-born contaminant known to display toxic effects on male reproduction, producing infertility in rats and humans. Using the rat as a model, we investigated whether or not testicular organogenesis, which, in the rat species, occurs during the second half of gestation, was at particular risk regarding 3-MCPD toxicity. Pregnant rats were given daily doses of 5, 10 or 25 mg/kg BW of 3-MCPD from days 11.5-18.5 postcoitum (dpc). On 19.5 dpc, testes were removed from fetuses for histological examination and testosterone analysis. Eight genes were selected among the differentiation markers of testicular cell lineages, and their expression was studied by RT-PCR. The levels of 3-MCPD and its main metabolite, beta-chlorolactic acid, were assayed in fetal tissues and dam plasma. Our results show a statistically significant decrease in the mean body weight gain of pregnant rats treated with 10 and 25 mg/kg BW of 3-MCPD. Fetal testes exposed to 3-MCPD exhibited normal histology and produced testosterone at levels that were similar to controls. In addition, 3-MCPD did not alter gene expression in the fetal testes. This lack of effect occurred under conditions where 3-MCPD and beta-chlorolactic acid were found to readily cross the placental barrier and diffuse throughout the fetal tissues. Our findings indicate that 3-MCPD has minimal effect on rat testicular organogenesis.

Evidence for similar expression of protein C inhibitor and the urokinase-type plasminogen activator system during mouse testis development.

Plasminogen activators (PAs) and their inhibitors (PAIs) are predicted to be involved in the restructuring events that characterize the testis throughout development. We here demonstrate that PAI-3 or protein C (PC) inhibitor (PCI) was expressed in a sexually dimorphic fashion during mouse gonad genesis, whereas PAI-1 and -2 exhibited no sex differences. PCI transcripts accumulated rapidly in the male gonad, from 12.5 d postcoitum onward. Western blot and immunohistochemistry analyses confirmed that male, but not female, fetal gonads produced PCI, and that Leydig cells are the site of PCI synthesis. The occurrence of testicular target proteases for PCI, i.e. PC and urokinase- and tissue-type PA, was further tracked using RT-PCR, plasminogen zymography, and/or immunohistochemistry. PC and tissue-type PA showed no variation between sexes. By contrast, urokinase-type PA and its receptor (uPAR; which dictates the site and extent of proteolysis) exhibited sex differences from 13.5-14.5 d postcoitum. At that time, uPAR expression was restricted to Leydig cells. At earlier ages, uPAR was uniformly and widely distributed in the gonads of both sexes. In adult testes, PCI and uPAR immunoreactivities were also present in Leydig cells. In addition, PCI, PC, and uPAR had a germinal origin. Collectively, these results support the hypothesis that PCI may contribute to proteolysis equilibrium within the testis by acting in tandem with urokinase in Leydig cells and with PC and/or urokinase in spermatogenic cells. It will be important to determine how this role is linked to the phenotype of sterility reported elsewhere in male mice with pci deleted.

Diethylstilbestrol inhibits the expression of the steroidogenic acute regulatory protein in mouse fetal testis.

This study investigated the early deleterious effects of an in-utero exposure to diethylstilbestrol (DES) on mouse testicular development. To that purpose, pregnant mice were injected daily with up to 100 microg/kg DES from 10.5 to 17.5 days postcoitum (dpc). At 18.5 dpc, testes were removed from fetuses for RNA (RT-PCR) and protein (Western blot, immunohistochemistry) analysis. Twenty-two genes were selected among which transcription factors, markers of differentiation of the different testicular cell lineages, steroidogenic enzymes and hormone receptors. The Steroidogenic Acute Regulatory (StAR) protein produced by the fetal Leydig cells was dramatically reduced in the DES-exposed testes. The P450c17 was the other gene modified following DES exposure. The alteration of these two genes is consistent with the decrease observed in the intratesticular testosterone levels, in the DES-exposed testes. Collectively, we demonstrated that DES did not alter testicular cell lineage specification but that it strongly inhibited the major function of the fetal Leydig cells.

Differential expression of tissue inhibitor of metalloproteinases type 1 (TIMP-1) during mouse gonad development.

In mammals, the gene Sry initiates signaling pathways triggering the differentiation of a testis from a sexually indifferent gonad. Assuming that these morphogenetic events may alter the proteolytic balance, the expression of matrix metalloproteinases (MMPs) and inhibitors (TIMPs) was investigated in gonads from 11.5 days postcoitum (dpc) onward, when testicular organogenesis occurs. Whereas selective MMPs and TIMPs (1-3) were detected in undifferentiated gonads (11.5 dpc) and in neonatal testes, a single TIMP (TIMP-1) was expressed in a sexually dimorphic manner from 12.5 dpc onward (i.e., after overt male gonad differentiation), demonstrated by using a semiquantitative reverse transcriptase-polymerase chain reaction and a Western blot analysis. To gain insight into the role of TIMP-1, the expression of gelatinases (mRNA levels and enzyme activity) was monitored. However, no sex differences could be evidenced, indicating that TIMP-1 was not inhibiting this class of MMPs during testis organogenesis. Apart from being an inhibitor of MMPs, TIMP-1 is known to display growth promoting activities. Of interest, testicular TIMP-1 (but not TIMP-2) levels were further enhanced up to 2 weeks of age, consistent with a role in the early postnatal testicular growth. We, therefore, established an organotypic culture system in which seminiferous cords may differentiate de novo and grow, depending on culture conditions. In that system and mimicking the in vivo situation, TIMP-1 immunolocalized strongly within the male gonadal territory and weakly in female gonads, in which no organization was evident. Experiments are now under way to determine to what extent TIMP-1 is a morphogenic gene involved in seminiferous cord formation and development.