Systematic review of hospital-wide complication registries.

BACKGROUND: An institutional registry covering all surgical specialties could be an implementation tool in quality benchmarking between hospitals and aid determination of their cost-effectiveness. The objective of this systematic literature review was to evaluate original articles on existing prospective surgical registries that can be used by single institutions across surgical specialties. METHOD: A systematic review of the literature using PRISMA guidelines was conducted for articles focusing on hospital-wide surgical registries. Single-specialty retrospective registries, non-defined outcome measures or system protocols, and studies not in English were excluded. RESULTS: Five articles were included for analysis. Evaluation of the articles revealed wide methodological heterogeneity in the classification and categorization of complications and data collection methods. CONCLUSION: Ideal surgical quality monitoring systems should be real-time, contain patient-related risk factors, and encompass all surgical specialties. At present, such institutional registries are rarely reported and no consensus exists on their standard definitions and methodology.

Magnetic resonance imaging of acute scrotum.

BACKGROUND AND AIMS: The aim of the pilot study was to evaluate the feasibility of dynamic contrast enhanced (CE)-magnetic resonance imaging (MRI) in the detection of testicular ischemia and its ability to differentiate testicle torsion from other causes of acute scrotum. MATERIAL AND METHODS: Seventeen boys or young men with an acute scrotum were included in the prospective study during the time period from October 2001 to December 2005. The median age of the patients was 16,4 (7-44) years. The duration of the symptoms preceding the MRI study varied from six hours to 30 days. The study protocol included physical examination by a surgeon, laboratory tests and Doppler ultrasound (DUS) and finally testicles were imaged by using a 1,5 T MRI scanner; T1-weighted and diffusion weighted images were produced. The gadolinium uptake, reported as the region of interest (ROI) perfusion values and presented as curves, was compared between the affected and contralateral testicle. In testicles with normal blood circulation the ROI values increased during the imaging time. Nine patients were operated on, because the spermatic cord torsion could not be excluded by clinical or DUS findings. RESULTS AND CONCLUSIONS: All the normal testicles gave increasing ROI values meanwhile all three testicles with torsion gave constantly low values referring to no perfusion. Other causes of acute scrotum, such as epididymitis and torsion of testicular appendage seemed to be related with normal perfusion. Dynamic CE-MRI seems to show reliably ischemia of testicle and thus it may be helpful in selecting patients with acute scrotum for urgent operation.

Magnetic resonance imaging of experimental testicular torsion.

We investigated the feasibility of contrast enhanced (CE)-dynamic magnetic resonance imaging (MRI) for the detection of testicular torsion induced hypoperfusion in an experimental rat model. Adult Sprague-Dawley rats were subjected to unilateral testicular torsion of 360 or 720 degrees. After 1 h, the tail veins of the anaesthetized rats were cannulated and T2 -, diffusion-weighted and T1-weighted CE-dynamic MRI were subsequently performed by a 1.5 T MRI scanner. On apparent diffusion coefficient (ADC) images, the region of interest values of the ischaemic and control testes was compared. From CE-dynamic MR images, the maximal slopes of contrast enhancement were calculated and compared. In testicular torsion of 360 degrees, the maximal slope of contrast enhancement was 0.072%/s vs. 0.47%/s in the contralateral control testis (p < 0.001). A torsion of 720 degrees diminished the slope of contrast enhancement to 0.046%/s vs. 0.37%/s in the contralateral testis (p < 0.001). Diminished blood flow during torsion also followed in decreased ADC values in both 360 degrees (12.4% decrease; p < 0.05) and 720 degrees (10.8% decrease; p < 0.001) of torsion. Torsion of the testis causes ipsilateral hypoperfusion and decreased gadolinium uptake in a rat model that can be easily detected and quantified by CE-dynamic MRI. In diffusion-weighted MRI images, acute hypoperfusion results in a slight decrease of ADC values. Our results suggest that CE-dynamic MRI in combination with diffusion-weighted MRI can be used to detect compromised blood flow due to acute testicular torsion.

Glove perforations in open and laparoscopic abdominal surgery: the feasibility of double gloving.

BACKGROUND AND AIMS: According to the traditional view, the glove protects the patient from the bacterial growth of the surgeons' hands and doing so prevents infections. Today, with growing incidences of HIV and Hepatitis B and C, surgical gloves are also important as protection for the surgeon. We compared the safety of double indicator gloves to standard single surgical gloves by investigating how often surgical gloves are punctured in laparoscopic and open gastrointestinal surgery. STUDY: As study material we gathered all gloves that had been used in gastrointestinal surgery in Satakunta Central Hospital during two months. 814 gloves from 274 operations were tested by using standardized water filling test method. RESULTS: In open surgery 67 gloves out of 694 had been punctured (9.6 percent). Puncture occurred in 22.5 percent of operations (53 out of 236). During open surgery 24 holes out of 35 were undetected with single gloves (69 percent). With double indicator gloves, only 3 out of 31 holes were unnoticed (10 percent). Long duration of operation increased the risk of puncture. In laparoscopic operations 4 gloves out of 120 had been perforated (3.3 percent). CONCLUSION: Double surgical gloves give markedly better protection in surgery. This is important especially in high risk operations.

Diffusion weighted magnetic resonance imaging of rat testes: a method for early detection of ischemia.

PURPOSE: We investigated the feasibility of diffusion weighted magnetic resonance imaging (MRI) for the early detection of ischemia in the testis. MATERIALS AND METHODS: Circulation to the right testis in Wistar rats was occluded by surgical ligation of the right funicle. The left side was sham operated and served as a control. The diffusion and T2-weighted MRI images of the 2 testes was performed postoperatively by a 1.5 Tesla MRI unit using a knee coil. On apparent diffusion coefficient images and T2-weighted images the region of interest values in the 2 testes were measured and statistically compared. RESULTS: At 1 hour after testicular funicle ligation the apparent diffusion coefficient was 18% lower in the ischemic than in the sham operated testis (p <0.0098). At 2 hours the difference was 20% (p <0.0017). In the signal-to-noise ratio on T2-weighted images there was no difference in the left and right testes. CONCLUSIONS: Altered diffusion occurs in an ischemic testis, which can be measured on MRI at 1.5 Tesla. Thus, diffusion-weighted MRI may be a helpful method for the differential diagnosis of acute testicular torsion.

The expression and regulation of Bcl-2-related ovarian killer (Bok) mRNA in the developing and adult rat testis.

OBJECTIVE: To study the role of Bcl-2-related ovarian killer (Bok) in the regulation of apoptosis in the testis of developing and adult rat. METHODS: Bok mRNA expression was analyzed by Northern hybridization before and after culturing rat seminiferous tubules in vitro. Seminiferous tubules were cultured with different hormones and growth factors. Changes in the expression level of Bok mRNA during testicular development was analyzed by Northern hybridization. Localization of Bok mRNA was verified by in situ hybridization. RESULTS: Bok mRNA was highly expressed in the rat testis, varying during development. Highest expression levels were found in immature rats. Highest hybridization intensity appeared to be in spermatogonia, pachytene spermatocytes and Sertoli cells. Treatment with FSH was able to inhibit spontaneous increase of Bok mRNA expression that occurred in the defined stages of the rat seminiferous epithelium. CONCLUSIONS: FSH protects germ cells from apoptosis and this protective effect may at least partly be due to the inhibition of Bok gene expression. The amount of apoptosis varies during testicular development and highest expression of Bok mRNA occurs at the time of apoptosis, suggesting a possible role for Bok in its regulation.

Cell death and survival during ovarian follicle development.

Mammalian germ cells arise in the yolk sac endoderm at the caudal aspect of the embryo and migrate to the mesodermally-derived gonadal ridge early in development. After the oogonia reach the gonadal ridge, the process of meiosis begins which coincides with the first major wave of apoptosis of female germ cells (Coucouvanis et al., 1993). Subsequently, oocytes progress to the dictyate stage of prophase I where they remain arrested until ovulation.

Hormonal and photoperiodic modulation of testicular mRNAs coding for inhibin/activin subunits and follistatin in Clethrionomys glareolus, Schreber.

Photoperiodic and hormonal modulation of mRNAs for testicular inhibin/activin subunits and follistatin were studied in a seasonally breeding rodent, the bank vole (Clethrionomys glareolus). Photoperiod-induced testicular regression had no effect on the relatively low steady-state levels of follistatin mRNA. Inhibin alpha (I alpha) and beta B (I beta B) mRNA levels were significantly higher in regressed than in active gonads, but inhibin beta A was undetectable. The effect of gonadotropin administration on testicular weight and mRNA concentrations differed between the sexually active and quiescent voles. Neither FSH (1.2 U/kg; s.c. for 5 days) nor hCG (600 IU/kg; s.c. for 5 days) affected testicular weight in sexually active voles, whereas both gonadotropins significantly increased testicular weight in photo-regressed individuals. FSH had no effect on I alpha or I beta B mRNA concentrations in the active testes, whereas excessive hCG challenge induced a decrease in the steady-state levels of these mRNAs. FSH induced an increase in I alpha mRNA concentrations in the regressed gonad, whereas both gonadotropins concomitantly down-regulated I beta B mRNA levels. In conclusion, the high expression of I alpha and I beta B mRNA in the regressed testis imply autocrine and paracrine roles for inhibin/activin in the quiescent gonad of seasonal breeders. Inhibin alpha-subunit expression is at least partly under the control of FSH in the bank vole testis.

Inhibin gene expression in a large cell calcifying Sertoli cell tumour and serum inhibin and activin levels.

Inhibin is a potential tumour suppressor gene product in the gonads. While inhibin gene products may have a role in tumourigenesis, serum inhibin levels can be used as a marker for ovarian tumours derived from granulosa cells. Tumours derived from Sertoli cells, testicular counterparts of granulosa cells, are rare. To assess whether inhibin could be used as a human Sertoli cell tumour marker, serum inhibin and activin levels and inhibin subunit mRNA expression in the testis were studied. Northern blot and in situ hybridization revealed abundant expression of inhibin alpha, beta A, and beta B subunit mRNAs in large cell calcifying Sertoli cell tumours found in a 12-year old boy with Carney complex. The tumours were multifocal and bilateral. Serum inhibin levels were clearly elevated at the time of the diagnosis, decreased by 50% after one of the testes was removed, and were low or undetectable after the second orchidectomy six weeks later. Activin was undetectable before the orchidectomies, while a low concentration of activin-A was measured after them. Follicle stimulating hormone (FSH) concentration increased from normal pubertal value to castration level as expected. Normal seminiferous tubules also showed inhibin subunit alpha and beta B mRNA expression, whereas inhibin beta A mRNA was expressed in normal Leydig cells. These data suggest that serum inhibin reflects Sertoli cell activity and can be used as a human tumour marker.

Expression and function of a proapoptotic Bcl-2 family member Bcl-XL/Bcl-2-associated death promoter (BAD) in rat ovary.

Bcl-2-related anti- and proapoptotic proteins are important in the decision step of the intracellular death program upstream from the caspase proteases. Targeted overexpression of Bcl-2 in ovarian somatic cells of transgenic mice leads to decreased apoptosis of granulosa cells and is associated with higher ovulation rate, increased litter size, and ovarian teratoma formation. The ability of exogenous Bcl-2 proteins to promote follicle cell survival suggests that the transgene can bind to endogenous ovarian Bcl-2 family members and modulate the intracellular apoptosis process in favor of cell survival. We used the yeast two-hybrid system to search for ovarian Bcl-2 interacting proteins. The screening of an ovarian fusion complementary DNA library yielded several clones encoding for the death agonist Bcl-XL/Bcl-2-associated death promoter (BAD). Dimerization of Bcl-2-related proteins mediated by the consensus Bcl-2 homology (BH) domains is essential for their apoptosis-regulating function. Consistent with these observations, yeast two-hybrid assays indicated that the interaction of Bcl-2 with BAD is dependent on both BH4 and BH2 domains of Bcl-2. Northern blot analysis showed a wide distribution of BAD messenger RNA (mRNA) in diverse tissues with highest levels in the lung, ovary, uterus, and brain. In situ hybridization analysis indicated BAD mRNA expression in granulosa cells of different sizes of follicles and also in the theca and interstitial cells. BAD mRNA was expressed in the ovaries between postnatal 15-27 days and did not alter during the developmentally occurring apoptosis found about postnatal day 18 when the first group of early antral follicles were formed. Similarly, BAD mRNA levels did not change during follicle atresia induced by estrogen withdrawal in immature rats. To study the role of BAD in the ovary, BAD complementary DNA was transfected into primary cultures of granulosa cells and in a gonadal tumor cell line. Overexpression of BAD induced apoptosis in both cell types, and the effect of BAD was reversed by a membrane-permeable caspase inhibitor, indicating that BAD induces apoptosis via the activation of caspase cysteine proteases. In summary, the death agonist BAD was identified as a Bcl-2-interacting protein in the ovary, and BAD mRNA is constitutively expressed in granulosa cells, suggesting that BAD is an essential part of the ovarian cell death process. Because BAD overexpression in granulosa cells leads to apoptosis, future studies on ovarian BAD binding proteins and hormonal regulation of the interactions among different Bcl-2 family members could provide a better understanding of the cellular mechanism of ovarian follicle atresia.

Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members.

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

Derivation of functional antagonists using N-terminal extracellular domain of gonadotropin and thyrotropin receptors.

Receptors for the glycoprotein hormones, LH/CG, FSH, and TSH, are a unique subclass of the seven-transmembrane, G protein-coupled proteins with a large N-terminal extracellular (ecto-) domain. Although ecto-domains of gonadotropin receptors confer ligand binding, expression of soluble binding proteins has been difficult. We fused the ecto-domains of LH or FSH receptors to the single-transmembrane domain of CD8 and found that hybrid proteins anchored on the cell surface retained high-affinity ligand binding. Inclusion of a junctional thrombin cleavage site in the hybrids allowed generation of soluble receptor fragments that interfered with gonadotropin binding to their receptors and blocked cAMP production stimulated by gonadotropins. Cross-linking analyses confirmed the formation of high molecular weight complexes between receptor ecto-domains and their specific ligands. A similar approach also generated a soluble TSH receptor fragment capable of blocking TSH-induced signal transduction. When administered to rats, the soluble FSH receptor fragment retarded testis growth and induced testis cell apoptosis. These findings demonstrate the feasibility of generating ligand-binding regions of glycoprotein hormone receptors to selectively neutralize actions of gonadotropins and TSH, thus allowing future design of novel contraceptives and management of different gonadal and thyroid dysfunction. The present study represents the first successful derivation of soluble, ligand-binding domains from glycoprotein hormone receptors as functional antagonists. Similar approaches could allow generation of ecto-domains of related receptors to neutralize actions of ligands or receptor antibodies and to facilitate structural-functional analysis.

Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11.

Apoptosis and survival of diverse cell types are under hormonal control, but intracellular mechanisms regulating cell death are unclear. The Bcl-2/Ced-9 family of proteins contains conserved Bcl-2 homology regions that mediate the formation of homo- or heterodimers important for enhancing or suppressing apoptosis. Unlike most other members of the Bcl-2 family, BAD (Bcl-xL/Bcl-2 associated death promoter), a death enhancer, has no C-terminal transmembrane domain for targeting to the outer mitochondrial membrane and nuclear envelope. We hypothesized that BAD, in addition to binding Bcl-xL and Bcl-2, may interact with proteins outside the Bcl-2 family. Using the yeast two-hybrid system to search for BAD-binding proteins in an ovarian fusion cDNA library, we identified multiple cDNA clones encoding different isoforms of 14-3-3, a group of evolutionally conserved proteins essential for signal transduction and cell cycle progression. Point mutation of BAD in one (S137A), but not the other (S113A), putative binding site found in diverse 14-3-3 interacting proteins abolished the interaction between BAD and 14-3-3 without affecting interactions between BAD and Bcl-2. Because the S137A BAD mutant presumably resembles an underphosphorylated form of BAD, we used this mutant to screen for additional BAD-interacting proteins in the yeast two-hybrid system. P11, a nerve growth factor-induced neurite extension factor and member of the calcium-binding S-100 protein family, interacted strongly with the mutant BAD but less effectively with the wild type protein. In Chinese hamster ovary (CHO) cells, transient expression of wild type BAD or its mutants increased apoptotic cell death, which was blocked by cotransfection with the baculovirus-derived cysteine protease inhibitor, P35. Cotransfection with 14-3-3 suppressed apoptosis induced by wild type or the S113A mutant BAD but not by the S137A mutant incapable of binding 14-3-3. Furthermore, cotransfection with P11 attenuated the proapoptotic effect of both wild type BAD and the S137A mutant. For both 14-3-3 and P11, direct binding to BAD was also demonstrated in vitro. These results suggest that both 14-3-3 and P11 may function as BAD-binding proteins to dampen its apoptotic activity. Because the 14-3-3 family of proteins could interact with key signaling proteins including Raf-1 kinase, protein kinase C, and phosphatidyl inositol 3 kinase, whereas P11 is an early response gene induced by the neuronal survival factor, nerve growth factor, the present findings suggest that BAD plays an important role in mediating communication between different signal transduction pathways regulated by hormonal signals and the apoptotic mechanism controlled by Bcl-2 family members.

Regulation of ovarian follicle atresia.

The majority of ovarian follicles undergo atresia, a hormonally controlled apoptotic process. Monitoring apoptotic DNA fragmentation provides a quantitative and sensitive endpoint to study the hormonal regulation of atresia in ovarian follicles. During follicle development, gonadotropins, together with local ovarian growth factors (IGF-I, EGF/TGF-alpha, basic FGF) and cytokine (interleukin-1 beta), as well as estrogens, activate different intracellular pathways to rescue follicles from apoptotic demise. In contrast, TNF-alpha, Fas ligand, presumably acting through receptors with a death domain, and androgens are atretogenic factors. These diverse hormonal signals probably converge on selective intracellular pathways (including genes of the bcl-2 and ICE families) to regulate apoptosis. With a constant loss of follicles from the original stockpile, the ovary provides a unique model for studying the hormonal regulation of apoptosis.

Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles.

In the mammalian ovary, only a small fraction of follicles fully mature and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian mediators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell types, and the apoptotic effect of TNF alpha is, partially, coupled to the sphingomyelin signaling pathway with ceramide as a second messenger. Because TNF alpha has been localized in the rat ovary, and TNF alpha treatment increases granulosa cell ceramide production, we studied the effect of treatment with TNF alpha and ceramide on follicle apoptosis. Immature rats were implanted with diethylstilbestrol to stimulate the development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treatments. During culture, spontaneous follicle apoptosis occurred (10-fold increase in DNA fragmentation), which was partially blocked by 100 ng/ml FSH (60% suppression). The effect of FSH was counteracted by TNF alpha in a dose-dependent manner, with the maximal effect at 100 ng/ml TNF alpha (90% reversal of FSH action). In situ analysis indicated that the granulosa cell is the follicle cell type undergoing DNA fragmentation. A membrane-permeable ceramide analog, C2-ceramide N-acetyl sphingosine, mimicked the effect of TNF alpha and was able to completely abolish the action of FSH at 50 microM. In contrast, another ceramide analog, C2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To study the mechanism of TNF alpha and ceramide action, the effect of sodium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-converting enzyme/ced-3-related cystine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with ATM (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating follicle apoptosis. Treatment with either TNF alpha or ceramide increased both basal and FSH-stimulated progesterone production by cultured follicles. Concomitant treatment by ATM did not alter the stimulatory effect of TNF alpha or ceramide on progesterone production, ruling out nonspecific toxic effect of the inhibitor and indicating that the apoptotic and steroidogenic pathways are independent. In summary, treatment with TNF alpha or its second messenger, ceramide, stimulates apoptosis of early antral follicles in culture, suggesting a potential role for TNF alpha as an intraovarian regulator of follicle atresia by acting through the ceramide signaling pathway.

Development of luteinizing hormone action in the perinatal rat ovary.

The expression of LH receptor (LHR) mRNA was studied in fetal rat gonads using polymerase chain reaction multiplication of reverse-transcribed mRNA. A primer pair corresponding to the extracellular domain of the receptor revealed the expression of LHR mRNA in fetal ovaries and testes as early as embryonic Day 13.5, the earliest age studied. The localization of LHR mRNA was examined in the perinatal rat ovary using in situ hybridization with two antisense probes, one encoding the extracellular and the other encoding the transmembrane domains of LHR. At the age of 4 days, only the extracellular LHR probe gave specific signal over ovarian stromal and follicular cells, excluding the ova. Three days later, similar distribution of specific hybridization was observed with both probes. In the 10- and 30-day-old rat ovaries, clear expression of LHR mRNA was found to be with both probes in theca cells. Gonadotropin-stimulated production of cAMP was studied in cultures of dispersed perinatal rat ovarian cells. When 5-day-old ovarian cells were cultured for 3 days in vitro and then stimulated by either hCG (0.1 mg/L) or FSH (1 mg/L) for 6 h, cAMP production was enhanced only in cells stimulated by FSH. In a similar experiment with 7-day-old ovarian cells, cAMP production was stimulated by both FSH and hCG. Stimulation with hCG (0.1 mg/L) during the 3-day culture caused homologous desensitization of cAMP production, but stimulation with FSH (0.1 mg/L) had no such effect. The desensitization of the LHR was also investigated by treating neonatal rats in vivo with a high dose of hCG (600 IU/kg BW s.c. as a single injection on Day 7) or with a dosage of recombinant human (rec) FSH on Days 3-9 (0.3 IU s.c twice daily). Thereafter, at the age of 10 days, the ovaries were incubated either with recFSH (200 IU/L) or hCG (CR-121; 0.1 mg/L) for 1 h. Homologous desensitization of cAMP production by hCG was observed, but the FSH-mediated cAMP production was not affected. The hCG-induced steroidogenesis (progesterone and testosterone production) was not desensitized. In conclusion, these findings indicate that 1) the expression of the mRNA encoding the extracellular domain of LHR, i.e., truncated receptor, occurs in fetal rat gonads as early as embryonic Day 13.5; 2) the expression of the truncated LHR mRNA occurs uniformly in the differentiating ovarian cells before the appearance of the functional theca cell layer; 3) full-length LHR message appears in the developing ovary concomitantly with appearance of differentiated theca cells; 4) homologous desensitization of cAMP output by hCG, without steroidogenic desensitization, is present in perinatal rat ovaries; and 5) no FSH-evoked desensitization of cAMP production occurs in perinatal ovaries.

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Sex-dependent expression of tenascin-C in the differentiating fetal rat testis and ovary.

The cellular mechanisms controlling sexual differentiation of fetal gonads are poorly understood. By examining the protein and mRNA expression of tenascin-C in correlation with the immunocytochemical detection of alpha-smooth muscle actin (alpha-SMA) and basement membrane heparan sulfate proteoglycan (HSPG) we demonstrate a clear-cut sex-and development-dependent expression pattern of tenascin-C in the rat testis, ovary and mesonephros. Immunocytochemistry and in situ hybridization of tenascin-C in 15-day-pc fetal testis and ovary showed protein and mRNA accumulation within the mesenchyme of the mesogonadal connection. In addition to the male and female mesonephros, some labeling could also be seen within the testicular tunica albuginea and intraovarian mesenchymal septa. In the 17-day-pc testis abundant accumulation of tenascin mRNA and protein appeared in the tunica and mediastinum testis, but not at all in the intratesticular mesenchyme. A similar pattern was still seen in the newborns where, however, a decrease in the anti-tenascin immunoreactivity of the tunica and mediastinum could be demonstrated. In contrast to the testis, expression of tenascin in 17-day-pc ovaries was widespread within the hilus and the entire intragonadal mesenchyme where it continued to accumulate also in newborns. Northern blot analysis of tenascin-C mRNAs showed one message of 8.0 kb in the 15-day-pc male and female gonads. An additional weak signal of 6.5 kb was seen in the female mesonephros. In the 18-day-pc testis, the 6.5-kb signal appeared stronger than the 8.0-kb signal. In contrast to the testis, the 6.5-kb message was weak in the developing ovary where the 8.0-kb signal had an intense peak on the day 18 pc. Further, in the ovary, mesenchymal accumulation of HSPG coincided with the spatial distribution of tenascin. In the testicular tunica and in the mesenchyme of the male and female genital ducts expression of tenascin was parallel with the differentiation of smooth muscle tissue, detected by labeling for alpha-SMA, which also indicated the tenascin-negative myoid cells of the testis. Our results indicate that tenascin expression in the fetal rat internal genitalia is involved in the differentiation of smooth muscle cells but not intratesticular myoid cells. In the ovarian mesenchyme, tenascin-C may have a specific function in the dynamic remodeling of the ovarian cords.

Localization of urokinase- and tissue-type plasminogen activator mRNAs in rat testes.

The expressions of urokinase (uPA) and tissue-type plasminogen activators (tPA) in different stages of the rat seminiferous epithelial cycle were analyzed by in situ and Northern hybridizations combined with zymographic analysis. Irradiated rat testes were used to assess the cell localization. Both of the plasminogen activators were expressed in a strictly stage specific manner. Maximal expression of uPA mRNA was seen in Sertoli cells during stages VII-VIII of the cycle. The same expression in the basal compartment of the tubules was detected at 7 days post-irradiation (p-i), during a selective reduction of spermatogonia and preleptotene spermatocytes. Levels of tPA mRNA started to accumulate in Sertoli cells at stage VIII and were high during stages IX-XII and detectable during stages XIII-XIV. At 26 days p-i, reduction of pachytene spermatocytes, which are shown to be immunoreactive for tPA, did not have an effect on tPA mRNA expression. Catalytic activities of uPA and tPA changed concomitantly to their RNA levels in different stages of the cycle. However, at 7 days p-i, uPA activity was decreased at stages VII-VIII of the cycle suggesting that germ cell Sertoli cell interaction is important for uPA activity.