Fine structure of muscle cells of the human testicular capsule: basis of testicular contractions.

Electron micrographs of human testicular capsule reveal large numbers of branching smooth muscle cells coursing through collagenous tissue of the tunica albuginea. These cells have subcellular morphology characteristic of smooth muscle cells, and they associate with one another through areas of close contact. These are the contractile cells responsible for spontaneous contractions of the human testicular capsule-contractions that may be important in transporting nonmotile sperm out of the testis.

Analysis of patients treated with living pig tissue for evidence of infection by porcine endogenous retroviruses.

The use of pigs as a source of cells and organs for transplantation has the potential to reduce the current chronic shortage of organs for the treatment of many end-stage diseases. The risk of transmission of infectious agents across the species barrier (zoonoses) has to be assessed. Many such agents can be eliminated from the pig herd. However, porcine endogenous retroviruses, which are carried within the pig genome, are not easily eliminated. They can infect primary and immortalized human cells in vitro, but to date no evidence for in vivo infection has been found in retrospective studies of humans exposed to viable porcine cells. Small-scale clinical trials using porcine cells for the treatment of Parkinson's and Huntington's disease are currently in progress. The prospective monitoring of these patients in conjunction with further research into the biology of this virus will help address safety issues.

In vivo analysis of porcine endogenous retrovirus expression in transgenic pigs.

Xenotransplantation offers a potential solution to the shortage of donor organs for allotransplantation. In vitro studies that demonstrate the transmission of porcine endogenous retroviruses (PERV) from porcine cells to human cells and cell lines have raised concerns regarding the potential transmission of PERV to both xenograft recipients and their contacts (1-4). While no evidence of infection has been detected in any patients who have been treated with a variety of different porcine tissues (5-8), two studies have shown that severe combined immunodeficient (SCID) mice can be infected by PERV after the transplantation of porcine islets (9-10). To further address the concerns of PERV, expression of this virus in tissues and serum from transgenic pigs that express human decay accelerating factor was investigated. Although viral mRNA expression was detected in a variety of tissues, no evidence of viral release was observed in any of the porcine tissues analyzed by transmission electron microscopy. Analysis of porcine serum using the product-based reverse transcriptase assay suggested that virions may be present in porcine serum from large white pigs. However, using methods based on those previously described by Wilson et al. (4), infectious virus was not detected when activated peripheral blood mononuclear cells from these pigs were cocultivated with human cells known to be permissive for PERV.

Tissue expression of human complement inhibitor, decay-accelerating factor, in transgenic pigs. A potential approach for preventing xenograft rejection.

Since complement-mediated hyperacute rejection of xenografts prevents the use of pigs as organ donors to man, the development of transgenic animals expressing species-specific complement inhibitors could provide a strategy for overcoming hyperacute rejection. The complement inhibitor, human decay-accelerating factor (hDAF), prevents the assembly of C3 and C5 convertases. In this article, the first histologic analysis of hDAF expression in pig tissues, specifically expression in endothelial cells of pigs transgenic for hDAF, is described. Twenty-seven transgenic pigs were categorized into 4 groups based on the expression patterns in endothelial, vascular smooth muscle, and squamous epithelial cells of skin biopsy specimens. Skin biopsy specimens permitted evaluation of the pigs without the need to kill them or to perform invasive procedures. Sixteen cases demonstrated endothelial cell staining. Complete necropsy evaluation, available in 14 of the 27 pigs, correlated with the skin biopsy specimen expression of hDAF. The immunoperoxidase data matched identically with the presence of the mRNA transcript in 25 of the 26 cases where RNA data were available. Also, the staining patterns of 6 transgenic pig founders and their 9 offspring (total of 9 founder-offspring pairs) correlated. Since transgenes are variably expressed in different cell types and since tissue lysates represent a melange of cell types, histologic evaluation for protein expression in tissues from transgenic animals will be critical if they are to be bred to become clinical organ donors. In addition to endothelial expression of hDAF, its expression on vascular smooth muscle cells may be important in preventing tissue damage when breaks in the endothelium occur.

Characterization of pigs transgenic for human decay-accelerating factor.

BACKGROUND: To prevent the central role played by complement activation in the hyperacute rejection of pig organs transplanted into primates, pigs transgenic for human decay-accelerating factor (HDAF) have recently been produced. The data presented here extend previous immunohistochemical findings by documenting the immunological characterization and the levels of expression of HDAF in these transgenic pigs. METHODS: Animals from 30 independently derived lines were included in this study. HDAF expression was characterized by immunoprecipitation and epitope mapping. Quantitative analysis was performed by radiometric assays followed by Scatchard analysis and by double-determinant radioimmunoassay. Deposition of iC3b on porcine aortic endothelial cells was determined by radioimmunoassay. DNA slot-blot analysis and densitometric scanning were used to evaluate HDAF transgene copy number. RESULTS: The integrity of HDAF expressed by these transgenic pigs could be demonstrated. HDAF was present in 72% of the organs analyzed, although considerable variation in expression occurred, both between animals and within the same pig. High levels of HDAF on porcine aortic endothelial cells resulted in iC3b deposition at levels as low as that detected on human endothelial cells. Twenty-six organs expressed levels of HDAF greater than those observed in the equivalent human tissue. HDAF expression did not correlate with the number of copies of the transgene incorporated into the porcine genome. CONCLUSIONS: Transgenic pigs, which express levels of functional HDAF even greater than those observed in humans, have successfully been produced. Pigs transgenic for human complement inhibiting molecules could represent a source of organs for future clinical xenotransplantation.

Orthotopic heart transplantation in a transgenic pig-to-primate model.

BACKGROUND: Previous studies demonstrated that hearts from transgenic pigs expressing human decay-accelerating factor (hDAF) were not hyperacutely rejected when transplanted heterotopically into the abdomen of cynomolgus monkeys. This study examines orthotopic transplantation of hDAF transgenic pig hearts into baboon recipients. METHODS: Orthotopic xenogeneic heart transplantation was performed using piglets, transgenic for hDAF, as donors. Ten baboons were used as recipients and were immunosuppressed with a combination of cyclophosphamide, cyclosporine, and steroids. RESULTS: Five grafts failed within 18 hr without any histological signs of hyperacute rejection. Pulmonary artery thrombosis induced by a size mismatch was observed in two of these animals. The other three recipients died because of failure to produce even a low cardiac output and/or dysrhythmia. The remaining five animals survived between four and nine days. One animal died of bronchopneumonia on day 4. Three xenografts stopped beating on day 5 due to acute vascular rejection. The longest survivor was killed on day 9 with a beating, histologically normal xenograft, because of pancytopenia. CONCLUSIONS: The results reported here demonstrate that hDAF transgenic pig hearts are not hyperacutely rejected when transplanted into baboon recipients. Orthotopically transplanted transgenic pig hearts are capable of maintaining cardiac output in baboons. An optimum immunosuppressive regimen is the subject of ongoing research.

Monitoring xenotransplant recipients for infection by PERV.

OBJECTIVES: Concerns have been raised over the possibility of transmission of porcine endogenous retrovirus (PERV) to porcine xenograft recipients. METHODS: To help assess this risk, diagnostic assays capable of detection of an active, latent or cleared PERV infection, and the presence of pig cell microchimerism have been developed by a number of groups. Retrospective studies of patients exposed to living pig tissues have been performed using these assays to look for evidence of cross species transmission. RESULTS: To date no evidence of PERV infection has been found in studies of humans exposed to pig tissues, despite evidence of long lived microchimerism. CONCLUSIONS: These data suggest that PERV infection has not occurred in a clinical setting. However, as infection has been seen in a small animal model further investigation of the risk from PERV is warranted.

Improved diagnostic accuracy by repetitive ultrasonic pregnancy testing in sheep.

Operator effects and instrument accuracy in using the Scanopreg ultrasonic pregnancy detector in sheep bred at synchronized estrus were studied in three experiments. In the first study, four operators tested the same 101 ewes at 60 and 80 days after breeding. The only significant difference among the four operators was that one operator consistently underestimated pregnancy. Operators did not differ in their diagnoses between days 60 and 80. In the second study, there were no differences between two operators who tested 239 ewes 90 days after breeding. In the third study, one operator tested 318 ewes 60, 70 and 90 days after breeding. The accuracy of diagnosis of pregnancy was at least 90% on each day tested; the corresponding diagnoses of nonpregnancy were 52, 76 and 79% correct. Some ewes that were initially diagnosed as nonpregnant were correctly recognized as pregnant when tested later than day 60. Most of the missed pregnancies were in ewes carrying a single lamb. A second Scanopreg test on day 90 of ewes not diagnosed pregnant on day 60 or 70 identified additional ewes as pregnant. Paired tests (days 70 and 90) recognized 99% of the ewes that eventually lambed.

Influence of body weight and number of inseminations on fertility of progestogen-treated ewe lambs raised in controlled environments.

The influence of body weight at breeding on reproductive response to one and two artificial inseminations (AI) of fresh extended semen was assessed in 195 crossbred ewe lambs selected for early breeding and compared with 159 adult ewes, all housed indoors in a controlled environment. In six trials, ewe lambs and adult ewes in progestogen-induced estrus were inseminated 55 to 57 h after sponge removal. One-half of the lambs and one-fourth of the adults received a second insemination at 60 h. Resultant reproductive performance of both groups indicated no advantage in a double insemination. Overall fertility, litter size and fecundity after one and two inseminations were 33%, 1.7 and .6 for ewe lambs and 68%, 2.4 and 1.6 for adult ewes, respectively. Embryonic mortality after the first 2 wk of pregnancy was estimated at 24% for ewe lambs and 9% for adults. The influence of body weight was analyzed by grouping the ewe lambs according to body weight at breeding. The lambs in group 1 weighed 30 to 35 kg; group 2, 36 to 40 kg; group 3, 41 to 45 kg and group 4, 46 to 50 kg. The proportion of ewes lambing in groups 1, 2, 3 and 4 was 16, 34, 39 and 48%, respectively. Corresponding litter sizes were 1.4, 1.6, 1.8 and 2.0. Fecundity increased (P less than 01) from .2 in group 1 to 1.0 in group 4. The results indicate that even when ewe lambs are bred by AI (eliminating a ram behavioral problem), sheep with heavier body weights produce more lambs per ewe bred.

Influence of PMSG and time of artificial insemination on fertility of progestogen-treated sheep in confinement.

The need for pregnant mates' serum gonadotropin (PMSG) in breeding confined sheep by artificial insemination (AI) at progestogen-synchronized estrus was assessed in 152 adult crossbred ewes brought into season by a controlled light regimen. One-half of the ewes received 500 IU PMSG after intravaginal progestogen treatment; all ewes were inseminated either 54, 57 or 60 h after sponge removal or at 54 and again at 60 h. Based on progesterone determinations 18 d after AI, conception rates with single insemination 54, 57, or 60 h and double insemination at 54 and 60 h were 76, 72, 47 and 72%, respectively, among ewes receiving PMSG, compared to 17, 22, 47 and 43%, respectively, among ewes not give PMSG (P less than .01) Lambing rates were higher (P less than .01) with PMSG (67, 67, 37 and 61%) than without PMSG (11, 11, 26 and 33%). While there was only a small increase (.06 less than P less than .05) in litter size with PMSG, fecundity decreased (P less than .01) from 1.4 to .3 when PMSG was not used. These data indicate that, even with controlled lighting to induce estrous activity, additional stimulation of ovulation by PMSG at progestogen-synchronized estrus is necessary for normal fertility when confined sheep are bred by AI.

Reproductive response of progestogen-treated sheep in confinement to a single and double insemination.

The reproductive responses to one and two inseminations of fresh extended semen were compared in crossbred sheep housed in confinement. In three trials, ewes in progestogen-induced estrus were inseminated 54 to 56 h after sponge removal. Half the ewes received a second insemination 4 or 5 h later. Resultant fertility, litter size and embryonic mortality indicated no advantage to a double insemination. Conception rates to one and two inseminations according to d 18 plasma progesterone levels were 66 and 63%, 85 and 86% and 74 and 80% in trials 1, 2 and 3, respectively. Corresponding lambing rates were 55 and 56%, 73 and 69% and 67 and 76%. Mean litter sizes were 2.1 and 2.5, while embryonic mortality after the first 2 wk of pregnancy was estimated at 13% for both groups. Single inseminations therefore simplify artificial insemination and double the number of ewes that can be inseminated/ejaculate.

Seasonal effects of PMSG and number of inseminations on fertility of progestogen-treated sheep.

The influence of pregnant mares' serum gonadotropin (PMSG) on reproductive performance of sheep bred by artificial insemination (AI) was studied in the anestrous and estrous seasons. Ewes were treated with progestogen-impregnated intravaginal sponges in June and October. One-half of the ewes in each trial received PMSG at sponge removal and were inseminated 55 h after sponge removal. One-half of the ewes in each treatment group were reinseminated 5 h later. Conception rates in June and October were 82 and 87% with PMSG and 18 and 48% without PMSG, respectively. The corresponding lambing rates were 60 and 74% with PMSG and 10 and 26% without PMSG. Litter size was unaffected by season or PMSG use. Embryonic mortality estimated over both trials was 22% after the first 2 wk of pregnancy with PMSG, but was 44% when PMSG treatment was omitted. Two inseminations were not superior to one. These data indicate that irrespective of season or double insemination, PMSG improves reproductive performance of ewes bred by AI at progestogen-synchronized estrus.

Influence of estradiol-17 beta on fertility in confined sheep inseminated with frozen semen.

Impaired sperm transport is believed to be a major cause of reduced fertiity in ewes inseminated with frozen semen. Because of suggestions that estradiol improves sperm transport, estradiol-17 beta was given to mature ewes at progestagen-induced estrus to test effects on fertility after artificial insemination with frozen semen. Conception rates, based on progesterone determinations made 18 days after insemination, were 43% for ewes injected with 50 microgram estradiol-17 beta and inseminated with frozen semsn, 52% for ewes inseminated with frozen semen but given no estradiol and 86% for ewes inseminated with fresh semen. Lambing rates were 6, 35 and 69%, respectively. The markedly greater difference between conception rates and lambing rates for the estradiol-treated ewes suggests that estradiol caused a substantial increase in the early embryonic mortality associated with the use of frozen semen.

Influence of sperm number and seminal plasma on fertility of progestagen-treated sheep in confinement.

Progestagen-impregnated vaginal sponges + PMSG were used to synchronize oestrus in crossbred adult ewes which were inseminated 56 h after sponge removal with 0.5 ml diluted semen containing 400, 200, 100, 50 or 25 x 10(6) spermatozoa per insemination. The diluent was skim milk-citrate or pooled seminal plasma. There was no difference in reproductive performance due to the insemination medium. Fertility (no. of ewes lambing) after insemination of 400 or 200 x 10(6) spermatozoa was 68% and was similar to that observed after natural service at progestagen-induced oestrus. When less than or equal to 100 x 10(6) spermatozoa were inseminated, fertility fell markedly and the number of lambs per ewe inseminated decreased. A decrease in litter size also occurred. The data indicate that insemination of 200 x 10(6) spermatozoa, i.e. less than 10% of the number in a single ram ejaculate, allows normal conception rates in progestagen-treated ewes.