Immunogenicity and contraceptive efficacy of plant-produced putative mouse-specific contraceptive peptides.

Rodent population control through contraception requires species-specific oral contraceptive vaccines. Therefore, in this study, we produced putative mouse-specific contraceptive peptides, mZP2 (from oocyte) and mIzumo1 (from sperm), in plants using Agrobacterium-mediated transient expression. Peptides were produced separately in Nicotiana benthamiana using constructs encoding antigens containing three copies of each peptide. We also determined the immunogenicity and contraceptive effects of the plant-produced antigens in female BALB/c mice. Mice immunized subcutaneously with a relatively low amount of antigen (5 µg/dose of each peptide in a mixture) showed systemic immune responses against mZP2-3 and mIzumo1-3 antigens. Moreover, the mean litter size of mice treated with the plant-produced antigens was reduced by 39% compared to that of the control mice. Notably, there was a significant negative correlation between the number of pups born and individual antibody levels against both antigens. Immunofluorescence assays demonstrated the binding of induced antibodies to the oocytes of BALB/c and wild-type mice in vivo and in vitro, respectively. Our study demonstrate the feasibility of producing small contraceptive peptides in plants that can be further used to develop oral contraceptive vaccines against mouse populations.

Oral and Subcutaneous Immunization with a Plant-Produced Mouse-Specific Zona Pellucida 3 Peptide Presented on Hepatitis B Core Antigen Virus-like Particles.

A short mouse-specific peptide from zona pellucida 3 (mZP3, amino acids 328-342) has been shown to be associated with antibody-mediated contraception. In this study, we investigated the production of mZP3 in the plant, as an orally applicable host, and examined the immunogenicity of this small peptide in the BALB/c mouse model. The mZP3 peptide was inserted into the major immunodominant region of the hepatitis B core antigen and was produced in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Soluble HBcAg-mZP3 accumulated at levels up to 2.63 mg/g leaf dry weight (LDW) containing ~172 µg/mg LDW mZP3 peptide. Sucrose gradient analysis and electron microscopy indicated the assembly of the HBcAg-mZP3 virus-like particles (VLPs) in the soluble protein fraction. Subcutaneously administered mZP3 peptide displayed on HBcAg VLPs was immunogenic in BALB/c mice at a relatively low dosage (5.5 µg mZP3 per dose) and led to the generation of mZP3-specific antibodies that bound to the native zona pellucida of wild mice. Oral delivery of dried leaves expressing HBcAg-mZP3 also elicited mZP3-specific serum IgG and mucosal IgA that cross-reacted with the zona pellucida of wild mice. According to these results, it is worthwhile to investigate the efficiency of plants producing HBcAg-mZP3 VLPs as immunogenic edible baits in reducing the fertility of wild mice through inducing antibodies that cross-react to the zona pellucida.

Plant-Produced Mouse-Specific Zona Pellucida 3 Peptide Induces Immune Responses in Mice.

Contraceptive vaccines are designed to stimulate autoimmune responses to molecules involved in the reproductive process. A mouse-specific peptide from zona pellucida 3 (mZP3) has been proposed as a target epitope. Here, we employed a plant expression system for the production of glycosylated mZP3 and evaluated the immunogenicity of plant-produced mZP3-based antigens in a female BALB/c mouse model. In the mZP3-1 antigen, mZP3 fused with a T-cell epitope of tetanus toxoid, a histidine tag, and a SEKDEL sequence. A fusion antigen (GFP-mZP3-1) and a polypeptide antigen containing three repeats of mZP3 (mZP3-3) were also examined. Glycosylation of mZP3 should be achieved by targeting proteins to the endoplasmic reticulum. Agrobacterium-mediated transient expression of antigens resulted in successful production of mZP3 in Nicotiana benthamiana. Compared with mZP3-1, GFP-mZP3-1 and mZP3-3 increased the production of the mZP3 peptide by more than 20 and 25 times, respectively. The glycosylation of the proteins was indicated by their size and their binding to a carbohydrate-binding protein. Both plant-produced GFP-mZP3-1 and mZP3-3 antigens were immunogenic in mice; however, mZP3-3 generated significantly higher levels of serum antibodies against mZP3. Induced antibodies recognized native zona pellucida of wild mouse, and specific binding of antibodies to the oocytes was observed in immunohistochemical studies. Therefore, these preliminary results indicated that the plants can be an efficient system for the production of immunogenic mZP3 peptide, which may affect the fertility of wild mice.

Two mouse lines selected for large litter size display different lifetime fecundities.

We recently described two outbred mouse lines that were selected for large litter size at first delivery. However, lifetime fecundity appears to be economically more important for the husbandry of many polytocous species for which mouse lines might serve as bona fide animal models (e.g. for pigs). In the present study, we compared the lifetime fecundities of two highly fertile mouse lines (FL1 and FL2: >20 offspring/litter at first delivery) with those of an unselected control line (ctrl) and two lines that were selected for high body weight (DU6) and high protein mass (DU6P) without selection pressure on fertility. We tested the hypothesis that selection for large litter size at first parturition would also increase lifetime fecundity in mice, and we observed very large differences between lines. Whereas FL1 and ctrl delivered up to nine and ten litters, none of the DU6 and DU6P females gave birth to more than five litters. In line with this observation, FL1 delivered the most pups per lifetime (85.7/female). FL2 females produced the largest average litter sizes (20.4 pups/litter) in the first four litters; however, they displayed a reduced number of litters. With the exception of ctrl, litter sizes declined from litter to litter. Repeated delivery of litters with high offspring numbers did not affect the general health of FL females. The presented data demonstrate that two biodiverse, highly fertile mouse lines selected for large litter size at first delivery show different lifetime reproductive fitness levels. Thus, these mouse lines might serve as valuable mouse models for investigating lifetime productivity and longevity in farm animals.

Altered testicular cell type composition in males of two outbred mouse lines selected for high fertility.

BACKGROUND: Recently we described two outbred mouse lines which have been selected for high fertility. These mouse models doubled the number of offspring per litter. OBJECTIVES: Although selected for a primarily female-trait of high fertility (increased litter size), we were interested whether also males of the fertility lines show differences within their reproductive organs. MATERIALS AND METHODS: We investigated males from two outbred mouse lines which have been selected for the phenotype "high fertility" for more than 170 generations. In the present study, we analysed the testicular cell type composition by flow cytometry. We further investigated the weights of reproductive organs, histomorphometry of testis as well as studied sperm motility parameters using a thermal stress assay as well as a sperm hyperactivation assay. RESULTS: Here, we describe that males of the fertility line (FL) 1 show an increased percentage of diploid cells within the testis. Flow cytometric analysis identified this enlarged cell population as Leydig cells. Testis weights were unaffected whereas the weights of seminal vesicles of FL1 and FL2 were increased compared to Ctrl bucks. FL2 males show decreased diameter of tubulus seminiferi and an enhanced spermatid/Sertoli cell index. Sperm motility parameters of FL1 and Ctrl males are initially indistinguishable but FL1 spermatozoa show a better performance in a thermal stress experiment over a 5 hours observation period. DISCUSSION: These data indicate that although selected for a primarily female-trait of high fertility also males from the fertility lines are effected by defined alterations in their reproductive organs. CONCLUSION: Some of these alterations are FL1-specific others are FL2-associated, indicating that different molecular strategies warrant the high-fertility phenotype on the female as well as on the male side.

Testicular transcriptional signatures associated with high fertility.

Factors of high fertility are poorly described. The majority of transgenic or knockout models with a reproductive phenotype are subfertile or infertile phenotypes. Few genotypes have been linked to improved reproductive performance (0.2%) or increased litter size (1%). In this study, we used a unique mouse model, fertility line FL1, selected for 'high fertility' for more than 170 generations. This strain has almost doubled the number of littermates as well as their total birth weight accompanied by an elevated ovulation rate and increased numbers of corpora lutea compared to a randomly mated and unselected control line (Ctrl). Here, we investigate whether the gonadal tissue of FL1 males are affected by 'co-evolution' after more than 40 years of female-focused selection. Using microarrays, we analysed the testicular transcriptome of the FL1 and Ctrl mice. These data were also compared with previously published female gonadal transcriptional alterations. We detected alterations in testicular gene expression, which are partly associated with female reproductive performance. Thus, female-focused selection for litter size has not only affected the female side, but also has been manifested in transcriptional alterations on the male gonadal organ. This suggests consequences for the entire mouse lines in the long run and emphasizes the perspective of inevitably considering both genders about mechanisms of high fertility.

Reproductive performance primarily depends on the female genotype in a two-factorial breeding experiment using high-fertility mouse lines.

Mouse models showing an improved fertility phenotype are barely described in the literature. In the present study, we further characterized two outbred mouse models that have been selected for the phenotype 'high fertility' for more than 177 generations (fertility lines (FL) 1 and 2). In order to delineate the impact of males and females on fertility parameters, we performed a two-factorial breeding experiment by mating males and females of the three different genotypes (FL1, FL2, unselected control (Ctrl)) in all 9 possible combinations. Reproductive performance, such as number of offspring per litter or total birth weight of the entire pup, mainly depends on the female genotype. Although the reproductive performance of FL1 and FL2 is very similar, their phenotypes differ. FL2 animals of both genders are larger compared to FL1 and control animals. Females of the control line delivered offspring earlier compared to FL1 and FL2 dams. Males of FL1 are the lightest and the only ones who gained weight during the two weeks mating period. To address whether this effect is correlated with differing serum androgen levels, we measured the concentrations of testosterone, dehydroepiandrosterone, 4-androstenedione, androstanediol and dihydrotestosterone in males of all three lines by GC-MS. We measured serum testosterone between 5.0 and 6.4 ng/mL, whereas the concentrations of the other androgens were at least one order of magnitude lower, with no significant differences between the lines. Our data indicate that reproductive outcome largely depends on the genotype of the female in a two-factorial breeding experiment and supports previous findings that the phenotype 'high fertility' is warranted by using different physiological strategies.

Polysialylation of NCAM correlates with onset and termination of seasonal spermatogenesis in roe deer.

Roe deer (Capreolus capreolus) are seasonal breeders and cyclic structural changes of roe bucks' testis come along with a totally arrested (winter) and a highly activated spermatogenesis (summer). For this reason, roe buck represents an interesting model to study general mechanisms of initiation and termination of spermatogenesis. We investigated if polysialic acid (polySia)-a linear homopolymer of α2,8-linked sialic acids, which could act as a negative regulator of cell-cell adhesion-might be involved in the activation and/or inactivation of spermatogenesis. To address this point, testis samples of adult male roe deer were collected at different time point of the year. Intriguingly, we observed that polySia attached to the neural cell adhesion molecule was enhanced during the onset of spermatogenesis in April. In addition, polySia was highly expressed in December. Predominantly, polySia was detectable between Sertoli cells and spermatogonia in the basal regions of testicular tubules and in the adluminal part of Sertoli cells. Interestingly, similar polySia distributions were observed during early testis development of other mammalians when gonocytes (pre-spermatogonia) and Sertoli cells represent the only cell populations in tubuli seminiferi. Thus, polySia is expressed during key steps of the "on/off mechanisms" of spermatogenesis and might represent one mediator of the interaction and communication between Sertoli cells and germ cell precursors.

Prenatal testosterone excess alters Sertoli and germ cell number and testicular FSH receptor expression in rams.

Exposure to excess testosterone (T) during fetal life has a profound impact on the metabolic and reproductive functions in the female's postnatal life. However, less is known about the effects of excess testosterone in males. The aim of the present study was to evaluate the impact (consequences) of an excess of T during fetal development on mature male testis. The testicular evaluation was by histological analysis and by determination of mRNA expression of the FSH receptor (FSH-R), transforming growth factor-ß type I receptor (TßR-I), and two members of the TGF-ß superfamily, transforming growth factor-ß3 (TGFß3) and anti-Müllerian hormone (AMH) in males born to mothers receiving an excess of T during pregnancy. At 42 wk of age, postpubertal males born to mothers treated with 30 mg of T propionate twice weekly from day 30 to 90, followed by 40 mg of T propionate from day 90 to 120 of pregnancy (T males), showed higher concentrations of FSH in response to a GnRH analog, a higher number of Sertoli cells/seminiferous tubule cross-section, and a lower number of germ cells/tubules (P < 0.05) than control males (C males) born to mothers treated with the vehicle. The mRNA expression of FSH-R and of TßR-I was higher in T males compared with C males (P < 0.05). Moreover, in T males, AMH expression level correlated negatively with the expression level of TGFß3. In C males, this latter correlation was not observed. These results suggest that prenatal exposure to an excess of T can negatively modify some histological and molecular characteristics of the mature testis.

Vascular endothelial growth factor A is a putative paracrine regulator in seasonally controlled spermatogenesis: insights from a ruminant model, the roe deer.

Vascular endothelial growth factor A (VEGFA) influences spermatogenesis, but its impact on seasonally regulated sperm production is still not fully understood. Thus, we investigated both expression levels and localisation of VEGFA and its receptors VEGFR1 and 2 in roe buck testis via real-time reverse transcription polymerase chain reaction and immunohistochemistry in relation to seasonal changes in the cellular composition of the testis. VEGFA was expressed by interstitial cells while its receptors were found on endothelial and perivascular cells. Inside the tubules, VEGFA was located in spermatogonia and spermatocytes, VEGFR1 was present on elongating spermatids and VEGFR2 on Sertoli cells. VEGFR1 mRNA was expressed tenfold lower than VEGFR2 and VEGF mRNAs. Relative VEGF and VEGFR2 expression (divided by the number of VEGFA and VEGFR2 expressing cells) showed an increase towards the rut (July/August) and a decrease thereafter. The results suggest involvement of VEGFA in the adjustment of vascular permeability as well as in spermiogenesis and the proliferation of spermatogonia.

Intestinal expression of TFF and related genes during postnatal development in a piglet probiotic trial.

Trefoil factor family (TFF) peptides provide protective and reparative effects by enhancing epithelial integrity and promoting mucosal restitution. TFF peptide expression is induced after mucosal damage. These processes are of central physiological relevance during the postnatal intestinal development and are strongly influenced during the weaning period. In piglets, weaning at early maturation stages frequently causes mucosal inflammation. The aim of this study was to evaluate postnatal intestinal TFF expression in a piglet probiotic trial. Low intestinal TFF2 expression was measured at early maturation stages. Weaning, however, was associated with a distinct response of increased TFF2 expression, indicating an important role in enhancing mucosal integrity. In the distal jejunum and ileum weaning could as well be associated with increased TFF3 mRNA levels. Differential TFF1 expression was not detected. Furthermore, TFF2 localization studies in different intestinal loci were performed by means of immunohistochemistry. Expression of selected genes (TGFA, EGFR, Cox-2) known to promote TFF signaling showed differential expression pattern as well, thereby providing further functional background. Furthermore, the expression patterns of EGFR observed in this study contribute to an advanced view of previous findings of EGFR regulation mainly obtained in rodents. An upregulated EGFR expression during early postnatal development suggests a local relevance to porcine intestinal maturation. However, a feed supplementation with the probiotic strain Enterococcus faecium did not influence TFF expression.

Reduced germ cell apoptosis during spermatogenesis in the teratospermic domestic cat.

Teratospermia (>60% morphologically abnormal sperm/ejaculate) is associated with increased sperm output in the domestic cat. The objective of this study was to determine whether increased sperm production in teratospermic donors was associated with disturbances in germ cell apoptosis, the usual mechanism for sperm cell elimination. Apoptosis was measured by evaluating DNA fragmentation, expression of Caspase-3, and anti-apoptosis repressor with caspase recruitment domain (ARC) in the testes of normospermic compared with teratospermic cats. Testes (n = 6 males/group) were obtained by bilateral castration and immediately fixed in Bouin solution. Results revealed that greater than 97% of cells labeled as DNA fragmented were tubular regardless of male type. Fewer (P < .05) apoptotic spermatogenic cells per tubule (0.52 +/- 0.11 cells/tubule, x +/- SEM) and per 100 Sertoli cells (3.79 cells/100 Sertoli cells) were observed in teratospermic compared with normospermic (1.25 +/- 0.36 cells/tubule and 6.44 cells/100 Sertoli cells) cats. Among the spermatogenic cells, fewer (P < .03) spermatocytes were positively labeled in teratospermic (0.3 +/- 0.07/tubule) compared with normospermic (0.83 +/- 0.28/tubule) counterparts. Neither donor type differed in Caspase-3 or ARC expression activity. However, each factor was both cell- and stage-specific in expression. Specifically, Caspase-3 was located in Sertoli cells, A-spermatogonia, and round spermatids at stage V. The ARC was found in primary spermatocytes at each stage of the spermatogenic cycle. These results demonstrate that the high incidence of morphologically abnormal sperm in teratospermic male cats is accompanied by a reduced elimination of defective spermatogenic cells via apoptosis.

Seasonal variations in the epididymis of the roe deer (Capreolus capreolus).

The roe deer shows a distinct seasonal breeding pattern accompanied with significant changes in testicular structure and function during the annual cycle. It serves as a uniquely well-characterized ruminant model system to investigate the regulation of testicular activity. However, data regarding the seasonal variations taking place in the epididymis of the roe buck are not available. Therefore, this study provides a detailed morphological description of the roe buck's epididymis (cell types and segments) and a qualitative as well as quantitative characterization of the seasonal changes in the different parts of the duct. For every second month of the complete seasonal cycle, five roe bucks were castrated (n=30). Seasonal changes in the cellular composition of the epididymis were studied by computer aided image analysis of histological preparations. With regard to morphological criteria we defined 6 segments (S) within the epididymis (ductuli efferentes and S1-5) during the active period. S1-3 are located in the caput, 4 represents the corpus and 5 the cauda epididymidis. The epithelium consists of principal cells, basal cells, macrophages, lymphocytes and apical cells, except for the ductuli efferentes (cuboidal epithelium composed of ciliated and unciliated cells) and S5 (no apical cells). The quantification of the three functional compartments within the organ (lumina, epithelium and interstitial tissue) revealed distinct and region-specific seasonal changes in the cellular composition of caput, corpus and cauda epididymidis. As expected, the duct with its surrounding tissue expands towards rutting season. In the caput this enlargement of the duct is primarily caused by the growth of the epithelial compartment, whereas in the cauda it is predominantly attributed to the dilatation of the lumen, which is filled with testicular and epididymal fluid and spermatozoa towards the rut. This leads to distinct changes in the tissue composition of samples taken from the three main regions of the epididymis at different times of the year. This morphometric study provides the prerequisite for investigations of regulation mechanisms in epididymis function.

Estrogens are involved in seasonal regulation of spermatogenesis and sperm maturation in roe deer (Capreolus capreolus).

Roe deer (a seasonal breeder, rut: July to August) is a well characterized model for studying the seasonal regulation of testicular activity. However, not much is known about the impact of estrogens on seasonally determined sperm production. We therefore explored the time and cell type specific expression of estrogen receptors and of enzymes involved in steroid biosynthesis in roe deer testicular parenchyma and in the epididymis. Every second month during the entire seasonal cycle five roe bucks were castrated (n=30). Estrogen receptor (ER) alpha, ERbeta and the enzymes P450Aromatase and P450C17 were localized immunohistochemically. The expression levels of ERalpha, ERbeta and P450Aromatase were evaluated by semi-quantitative Western blot. Contrary to the enzyme required for androgen production (P450C17), which is expectedly located only in the Leydig cells and shows an expression increase towards rutting season, a seasonal expression difference of the enzyme required for the conversion into oestradiol (P450Aromatase) is visible only in the epididymis. In the testis, ERalpha expression shows a striking dependency on tubular cell composition, and the single cell expression activity increases towards rut. This implicates that estrogens are directly involved in the regulation of spermatogenesis in the roe buck. In the epididymis, expression of ERalpha is seasonally determined particularly in the ductuli efferentes. ERbeta was detected throughout the year with no distinct dependency on season or the stages of germinative epithelium cycle. We conclude that estrogens in the roe buck influence the seasonally determined sperm production predominantly by the regulated expression of ERalpha.

Impact of Bacillus thuringiensis toxin Cry1Ab on rumen epithelial cells (REC) - a new in vitro model for safety assessment of recombinant food compounds.

The growing use of genetically modified crops necessitates viable screening methods for safety evaluation of recombinant feed, particularly for ruminants. A new sheep rumen epithelial cell culture is introduced as an in vitro cell system for safety evaluation especially focussing on feed and food compounds. We used lactate dehydrogenase (LDH) release, WST-1 conversion, ATP content and caspase 3/7 activity to evaluate cytotoxicity of Cry1Ab, one of the newly expressed Bt-proteins in transgene maize. The results were compared to the effects of valinomycin, a potassium ionophore known to induce cytotoxic effects on a wide range of cells. Whereas no toxicity of Cry1Ab was observed in short as well as in long term experiments, even at non-physiological high concentrations, exposure to valinomycin induced apoptosis and a significant response of all viability parameters after a number of hours. The ATP content and the WST-1 conversion reflecting the energy metabolism of the cells appear to be more sensitive indicators of valinomycin toxicity than the LDH release, a parameter which reflects the membrane integrity. This study presents an in vitro model system, that may be useful as a supplementary tool in toxicity screening before testing substances on animals in vivo.

Apoptosis is not the cause of seasonal testicular involution in roe deer.

Apoptosis is involved in the regulation of spermatogenesis. The involution of testes in seasonal breeders might be expected to involve enhanced apoptotic cell elimination. We have compared seasonally changing testicular apoptosis in roe deer with that in non-seasonally breeding cattle. Apoptotic cells were detected as TUNEL-positive cells by both flow-cytometric analysis and in situ localisation of fragmented DNA in tissue sections. Apoptosis-induced DNA fragments were also assessed by enzyme-linked immunosorbent assay (ELISA) in homogenised testicular parenchyma. As expected, the testis mass and the percentage of haploid cells in roe deer showed a seasonal pattern with a significant maximum during the rut (August), whereas no annual variation of these parameters was found in bulls. All three methods for determining apoptosis showed similar findings. Roe deer exhibited significant seasonal fluctuation of total apoptotic activity (ELISA, apoptotic cells per tubule cross section) with a maximum during the breeding season. However, the seasonal differences in the number of apoptotic cells corresponded to the variable total numbers of spermatogonia and spermatocytes per tubule cross section. Thus, the percentages of TUNEL-positive cells related to the combined number of both germ cell types showed no seasonal variance, as confirmed by percentages of apoptotic cells analysed flow-cytometrically. The maximum level of apoptosis during the rut in roe deer was similar to the values obtained during the invariably high spermatogenic activity in cattle. These results suggest that, in roe deer, apoptosis is not the cause of the seasonal involution of testes.

Seasonally activated spermatogenesis is correlated with increased testicular production of testosterone and epidermal growth factor in mink (Mustela vison).

Seasonal changes in spermatogenesis were studied with respect to testicular production of both testosterone and epidermal growth factor (EGF) in mink. The testes were collected in November (n = 15; testis recrudescence), February (n = 15; before breeding season), March (n = 14; breeding season), and May (n = 11; testis involution) and the following parameters of testicular activity were quantified: testicular mass, number of testicular spermatozoa, percentages of haploid, diploid, and tetraploid (G2/M-phase) cells and content of testosterone and EGF. The growth factor was immunohistochemically localized in the parenchyma. Testis mass, spermatogenic activity, and the production of both testosterone and EGF were maximal in March, but were not significantly different from the levels in February. The correlation between testis weight and sperm per testis was r = 0.825 (P < 0.001). Testosterone and EGF levels were correlated to each other (r = 0.78; P < 0.001) and had significant positive correlations to testis mass, number of sperm and proportion of haploid cells; and negative correlations to percentages of mitotic cells. EGF was localized in interstitial cells and in the luminal region of seminiferous tubules, where it occurred during the last steps of spermiogenesis. We inferred that intensified seasonal spermatogenesis was stimulated by testosterone and by autocrine/paracrine effects of EGF.

The roe deer as a model for studying seasonal regulation of testis function.

In mature male seasonal breeders, the circannual cycles of testicular growth and involution involve significant changes in structure and function of both the tubular and interstitial testicular compartment. Roe deer (Capreolus capreolus) are seasonal breeders with a short defined rutting season from mid-July to mid-August and represent a unique non-rodent model for studying testicular functions during the course of a complete reproductive cycle with naturally changing photoperiod. Germ cells and Sertoli cells of the seminiferous tubules and the interstitial Leydig cells all display significant morphological and physiological alterations during the seasonal changes. In contrast to the germ cell population, Sertoli and Leydig cells persist as a numerically constant cell population in the roe deer testis. This report emphasizes the intricate relationship between seasonal cellular differentiation, intratesticular growth factor networks and their impact on the functional dynamics during the seasonal changes in roe deer testis.

Seasonal expression of INSL3 and Lgr8/Insl3 receptor transcripts indicates variable differentiation of Leydig cells in the roe deer testis.

Roe deer are seasonal breeders and show cyclic variation in testicular volume and cellular differentiation within the tubular and interstitial testis compartment. We have employed the roe deer as a model to elucidate the expression of the postpubertal Leydig cell marker INSL3 during seasonal changes in Leydig cell differentiation. Roe deer testis and serum samples were collected bimonthly throughout the complete reproductive cycle. Peak levels of testicular Insl3 mRNA and INSL3 immunoprotein were detected well before the onset of rut in April and coincided with the highest percentage of INSL3-positive cell number/square millimeter of testicular interstitial area. During the winter (December, February), roe deer INSL3 was exclusively detected in a subpopulation of alpha-actin-negative, spindle-shaped peritubular cells. Concordant with the increase in INSL3 production in April and 1 mo after the reported LH peak, a sharp increase in serum testosterone concentrations was observed. High serum testosterone concentrations coincided with the presence of detectable 17alpha-hydroxylase, mRNA and protein, in Leydig cells. Upregulation of INSL3 production in spring appeared to reflect LH-dependent differentiation of Leydig cells. The considerable changes in percentage of INSL3 immunopositive cells within the numerically constant interstitial cell population indicated cyclic seasonal de- and redifferentiation of Leydig cells. A complex functional role of the INSL3/LGR8 ligand-receptor system in the roe deer testis was suggested by the detection of specific hybridization signals for roe deer Lgr8 transcripts in Sertoli cells of the roe deer testis.

Continuous addition of adenosine with a micropump system improves warm whole blood cardioplegia.

BACKGROUND: Micropump additive systems allow for continuous modification of cardioplegia composition during heart surgery. Although the use of such systems in warm heart surgery is theoretically desirable, the role of the systems has been clinically limited by coronary vasoreactivity with higher potassium concentration and unreliable mechanical arrest at lower potassium concentration. Adenosine, a potent coronary vasodilator and arresting agent, has the potential to reduce the potassium concentration required for arrest and to improve distribution of cardioplegia. However, clinical use of adenosine has been limited by a short half-life in blood and difficulty in titrating the dose. This study tested the hypothesis that continuous addition of adenosine with an in-line linear micropump system would facilitate whole blood hyperkalemic perfusion for cardiac surgery. METHODS: Canine hearts (n = 9) were randomized to 20 minutes of arrest with whole blood cardioplegia or cardioplegia with adenosine at either low (0.5 M) or high (8 M) concentration. Potassium was supplemented at an arresting dose (24 mEq/L) for 5 minutes and then at a maintenance dose (6 mEq/L) for an additional 15 minutes. Coronary flow was held constant (4 mL/kg per minute), and aortic root pressure was measured. Myocardial performance was assessed by measurement of the end-diastolic pressure to stroke volume relationship at constant afterload. Myocardial tissue perfusion was evaluated with colored microspheres. RESULTS: During the initial period of high-concentration potassium arrest, coronary resistance rose progressively regardless of adenosine addition. Coronary resistance remained elevated during the period of low potassium perfusion, except when high-concentration adenosine was added. With addition of 8 M adenosine, coronary resistance returned to baseline, and left ventricular endocardial perfusion was augmented. Electromechanical quiescence improved with adenosine perfusion and was complete with high-dose adenosine addition. Function was preserved in all hearts. CONCLUSION: Use of a modern micropump system allowed for continuous addition of adenosine and potassium to whole blood cardioplegia. Adenosine minimized potassium-induced coronary vasoconstriction and improved endocardial perfusion and mechanical quiescence. These findings supported addition of adenosine to the perfusate during warm whole blood cardioplegia.