David M. de Avila

Spermatogenesis and Germ Cell Transgene Expression in Xenografted Bovine Testicular Tissue

Abstract The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the β-galactosidase gene in neonatal bovine testicular tissue ectopically xenografted onto the backs of recipient nude mice. Bull testicular tissue from 4-wk donor calves, which contains a germ cell population consisting solely of gonocytes or undifferentiated spermatogonia, was grafted onto the backs of castrated adult recipient nude mice. Testicular grafts significantly increased in weight throughout the grafting period and the timing of germ cell differentiation in grafted tissue was consistent with postnatal testis development in vivo relative to the bull. Seminiferous tubule diameter also significantly increased with advancing time after grafting. At 1 wk after grafting, gonocytes in the seminiferous cords completed migration to the basement membrane and differentiated germ cell types could be observed 24 wk after grafting. The presence of elongating spermatids at 24 wk confirmed that germ cell differentiation occurred in the bovine tissue. Leydig cells in the grafted bovine tissue were also capable of producing testosterone in the castrated recipient mice from 4 wk to 24 wk after grafting at concentrations that were similar to levels in intact, nongrafted control mice. The testicular tissue that had been electroporated with a β-galactosidase expression vector showed tubule-specific transgene expression 24 wk after grafting. Histological analysis showed that transgene expression was present in both Sertoli and differentiated germ cells but not in interstitial cells. The system reported here has the potential to be used for generation of transgenic bovine spermatozoa.

Testis Tissue Explant Culture Supports Survival and Proliferation of Bovine Spermatogonial Stem Cells

Abstract The present study was designed to evaluate the survival and proliferation of bovine spermatogonial stem cells in an explant culture system over a 2-wk period. Explants of calf testicular parenchyma were placed on 0.45-μm pore membranes in culture and maintained for 1–2 wk. Histological examinations of fresh (t0) and cultured tissues revealed morphologically normal seminiferous tubules. Germ cell numbers/tubule increased (P ≤ 0.05) during culture when compared with t0, yet germ cell differentiation was not observed. Testosterone was present in medium throughout the culture period, indicating functional Leydig cells. Sertoli, spermatogonial, and spermatogonial stem cell viability was evaluated by reverse transcription-polymerase chain reaction for cell-specific gene expression of stem cell factor, protein gene product 9.5, and glial cell line-derived neurotrophic factor family receptor-α1, respectively. Results demonstrated the expression of all genes at t0, 1 wk, and 2 wk of culture. Single-cell suspensions were prepared from the testicular tissues at t0 and during culture and transplanted into nude mouse testes to investigate spermatogonial stem cell viability. One month after transplantation, colonies of round bovine cells were identified in all mouse testes analyzed, indicating survival of spermatogonial stem cells. The average number of resulting colonies in recipient testes was significantly (P ≤ 0.05) higher following 1 wk of culture compared with t0 and was numerically higher at 2 wk of culture compared with t0. This increase in colony numbers over time in culture indicates spermatogonial stem cell proliferation in vitro. This explant culture system appears to provide an environment that supports survival and proliferation of bovine spermatogonial stem cells.

Immunization of Male Mice with Luteinizing Hormone-Releasing Hormone Fusion Proteins Reduces Testicular and Accessory Sex Gland Function

Abstract Genes for ovalbumin-luteinizing hormone-releasing hormone 7 (LHRH-7) and thioredoxin-LHRH-7 fusion proteins (containing seven LHRH inserts) were constructed by cassette and mismatch mutagenesis and expressed in Escherichia coli. In experiment 1, 10 μg of either ovalbumin-LHRH-7 or thioredoxin-LHRH-7 were suspended in Z-max adjuvant and injected three times at 4-wk intervals into postpubertal male BALB/c mice. In experiment 2, the fusion proteins were suspended in Immumax adjuvant and administered in equimolar quantities (0.4 nmol per injection) to postpubertal male BALB/c mice. In addition to injection of these two proteins alone, the proteins were also administered in different sequences or together in a mixture. Both LHRH fusion proteins induced significant antibody titers, which resulted in a significant decrease in vesicular gland and anterior prostate weight (measure of biological response) in both experiments. Vesicular gland and anterior prostate weight and LHRH antibody titers were significantly correlated in experiments 1 (r = −0.64) and 2 (r = −0.53). Percentage of animals responding to treatment varied from 40–60% in experiment 1 and from 11–89% in experiment 2, with the highest responses in treatments that used a combination of both fusion proteins. The variation in responders and nonresponders was evaluated by estimating antibody KD from displacement curves. Part, but not all, of the high antibody nonresponders can be explained by antibody affinity.

CpG motif-based adjuvant as a replacement for Freund's complete adjuvant in a recombinant LHRH vaccine

This study compared: (1) Freunds complete adjuvant and CpG oligodeoxynucleotide (ODN) 2006 in water-in-oil emulsion as adjuvants; and (2) increasing doses of a recombinant ovalbumin-LHRH (ova-LHRH) fusion protein as an antigen for a contraceptive vaccine. Treatment groups (n=8 heifers/group) were: one untreated control group; five groups receiving CpG ODN with different doses of ova-LHRH (1.5; 2.3; 3.4; 5.1; and 7.6 mg); and one group receiving 3.4 mg ova-LHRH in Freunds. Heifers were immunized at weeks 0 and 14. All vaccine treatments caused gonadal regression and estrus suppression. CpG ODN is a suitable replacement for Freunds for LHRH immunization.

Noninvasive monitoring of adrenocortical function in captive jaguars (Panthera onca).

Jaguars are threatened with extinction throughout their range. A sustainable captive population can serve as a hedge against extinction, but only if they are healthy and reproduce. Understanding how jaguars respond to stressors may help improve the captive environment and enhance their wellbeing. Thus, our objectives were to: (1) conduct an adrenocorticotrophic hormone (ACTH) challenge to validate a cortisol radioimmunoassay (RIA) for noninvasive monitoring of adrenocortical function in jaguars; (2) investigate the relationship between fecal corticoid (FCM) and androgen metabolite (FAM) concentrations in males during the ACTH challenge; and (3) establish a range of physiological concentrations of FCMs for the proposed protocol. Seven jaguars (3 M, 4 F) received 500 IU/animal of ACTH. Pre- and post-ACTH fecal samples were assayed for corticoid (M and F) and androgen metabolites (M) by RIA. Concentrations of FCMs increased (P80.01) after ACTH injection (pre-ACTH: 0.90 ± 0.12 µg/g dry feces; post-ACTH: 2.55 ± 0.25 µg/g). Considering pre- and post-ACTH samples, FCM concentrations were higher (P80.01) in males (2.15 ± 0.20 µg/g) than in females (1.30 ± 0.20 µg/g), but the magnitude of the response to ACTH was comparable (P>0.05) between genders. After ACTH injection, FAMs increased in two (of 3) males; in one male, FCMs and FAMs were positively correlated (0.60; P80.01). Excretion of FCMs was assessed in 16 jaguars (7 M, 9 F) and found to be highly variable (range, 80.11-1.56 µg/g). In conclusion, this study presents a cortisol RIA for monitoring adrenocortical function in jaguars noninvasively.

Effects of a gonadotropin-releasing hormone vaccine on ovarian cyclicity and uterine morphology of an Asian elephant (Elephas maximus).

This report describes the successful use of a gonadotropin-releasing hormone (GnRH) vaccine to suppress ovarian steroidogenic activity and to treat hemorrhage and anemia associated with reproductive tract pathology in a 59-year-old Asian elephant (Elephas maximus). The Repro-BLOC GnRH vaccine was administered subcutaneously as a series of 4 boosters of increasing dose from 3 to 30 mg of recombinant ovalbumin-GnRH fusion protein given at variable intervals after initial vaccination with 3 mg protein. Efficacy was confirmed over a year after initial vaccination based on complete ovarian cycle suppression determined by serum progestagen analyses. Estrous cycle suppression was associated with a significant increase in GnRH antibody binding and subsequent decrease in serum luteinizing hormone and follicle-stimulating hormone concentrations. Ultrasonographic examinations of the reproductive tract documented a reduction in uterine size and vascularity after immunization. The hematocrit level normalized soon after the initial intrauterine hemorrhage, and no recurrence of anemia has been detected. No substantive adverse effects were associated with GnRH vaccination. The results indicate that GnRH vaccination in elephants shows potential for contraception and management of uterine pathology in older elephants.

Immunization Against Luteinizing Hormone-Releasing Hormone Fusion Proteins Does Not Decrease Prostate Cancer in the Transgenic Adenocarcinoma Mouse Prostate Model

This study was undertaken to test the effect of immunization against luteinizing hormone-releasing hormone (LHRH) fusion proteins on the development and progression of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Two LHRH fusion proteins, ovalbumin with seven LHRH peptides (OV-LHRH-7), and thioredoxin with seven LHRH peptides (TH-LHRH-7) were used in a cocktail vaccine. Two groups of male TRAMP mice were immunized with the cocktail. Primary immunizations were at either 4 or 8 weeks of age. LHRH immunized mice (n = 19) were compared with castrated (n = 19) and intact mice (n = 18) for testosterone concentration, tumor weight, and lifespan. Immunization against LHRH in the TRAMP mice resulted in significant production of antibodies to LHRH compared with surgically castrated and intact control mice. Testicular weight was significantly reduced in the LHRH immunized groups compared with intact control mice. Serum testosterone was reduced (P < 0.05) in the immunized mice compared with intact control mice and was not different from that of castrated mice (P > 0.05). Tumor weight was variable and inconsistent throughout all treatment groups. Lifespan was not increased by immunization against LHRH or castration. Intact control mice (lived the longest (227 ± 11 days), whereas immunized mice lived 206 ± 11 days and castrated mice lived 213 ± 13 days. Tumors from immunized TRAMP mice appeared more aggressive than tumors of castrated and intact mice, as demonstrated by 35% expression of gross lung tumors in the immunized mice whereas none were observed in the castrated or intact TRAMP mice. Prostate cancer is initially dependent upon androgens for growth and development, but cells have the ability to escape androgen dependence and progress to an androgen independent state, which was evident in this study. The TRAMP mouse model immunized against LHRH may have utility in future studies and treatments of the androgen independent prostate cancer.