Ann Hahnel

Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro

Various times of in vitro maturation of oocytes, and three methods of separating spermatozoa from frozen-thawed semen (Percoll density-gradient centrifugation, swim-up, and glass-wool filtration), were compared for their effects on goat embryo production in vitro. Cumulus-oocyte-complexes (COCs) from abattoir ovaries were matured in M199 supplemented with 10% fetal calf serum and hormones. In Experiment 1, COCs were fixed at 4 h intervals from 0 to 27 h of culture to assess oocyte nuclear maturation. A higher proportion cultured for 27 h than for 24 h were in Metaphase II (27/37, 73% vs. 18/33, 55%, P < 0.05). In Experiment 2, the effects of separation methods on total numbers and numbers of membrane-intact spermatozoa, and the acrosome reaction were compared. Total numbers after Percoll density-gradient centrifugation were approximately 4 times higher than after swim-up and approximately 2 times higher than after glass-wool filtration (P < 0.001). Progression of the acrosome reaction was not affected differentially. In Experiments 3 and 4, after 27 h of culture the COCs were inseminated with sperm isolated by the three methods. In Experiment 3, presumptive zygotes were examined for pronucleus (PN) formation at 6, 12, 18 and 24 h post-insemination. At 12 h, male PN formation rate from Percoll-treated spermatozoa was higher than from sperm subjected to swim-up and glass-wool treatments (20/37, 54% vs. 6/37, 16% and 6/38, 16%, respectively; P < 0.001). In Experiment 4, embryos were compared for cleavage at 48 h and development into blastocysts, hatching rates and cell number at 192 h. The rates of cleavage and blastocyst formation in the Percoll-treated group were higher (P < 0.05) than in the swim-up and glass-wool groups (62% and 18% vs. 50% and 11%, and 45% and 8%, respectively). Similarly, the mean cell number in the Percoll group was higher (P < 0.05) than in the swim-up and glass-wool groups (167 +/- 5 vs. 149 +/- 4 and 126 +/- 4, respectively). We conclude that Percoll density-gradient centrifugation is superior to the other two methods for separating goat spermatozoa from frozen-thawed semen in preparation for IVF.

An analysis of the uterine lymphocyte-derived hybridoma cell line GWM 1-2 for expression of receptors for estrogen, progesterone and interleukin 2

Characterisation of murine hybridoma cell lines derived from the fusion of lymphocytes migrating from explant cultures of early, pregnancy-associated metrial glands (days 6-8 of gestation) to SP 2/0 cells, has been extended (van den Heuvel et al., J. Reprod. Immunol., 27 (1994) 13-36). These hybridomas have been grown in culture for over 2 years and are thought to represent the only immortalized lines of murine pregnancy-associated, uterine natural killer (uNK) cells. Previous studies had shown that these hybridomas, known as GWM cells, lack uNK cell surface markers, but share with uNK cells the expression of the lytic protein perforin and the ability to lyse YAC cells, a natural killer cell target (van den Heuvel et al., J. Reprod. Immunol., 27 (1994) 13-36). We report here, the evaluation of the transcription and expression of genes encoding the estrogen receptor (ER), the progesterone receptor (PR) and the interleukin 2 receptor complex (IL 2R alpha, beta and gamma) by uNK cells at day 8 of gestation and by GWM 1-2 cells and SP 2/0 cells. Our investigations indicate that expression of these genes divides day 8 uNK cells into subsets, with the predominant population being ER+, PR-, IL 2R alpha +, IL 2R beta + and IL 2R gamma +. Like day 8 uNK cells, most GWM 1-2 cells expressed all three chains of the IL 2R complex. In addition, GWM 1-2 cells expressed the ER but the PR was not detected on this cell line. Only the IL 2R alpha was detected on the SP 2/0 myeloma cell line. These studies further validate the use of GWM hybridomas as models for pregnancy-associated uNK cells.

Uterine Natural Killer Cells Do Not Require Interleukin-2 for Their Differentiation or Maturation

PROBLEM: Natural Killer lymphocytes (NK cells) from the pregnant uterus and from other tissues in pregnant and nonpregnant mammals can be stimulated by interleukin‐2 (IL‐2) during culture to become Lymphokine Activated Killer (LAK) cells. The susceptibility of cultured trophoblast cells to lysis by LAK cells raises the enigma of why uterine (u) NK cells that are characterized by morphology and by surface phenotyping as “activated,” and thus potentially damaging to the placenta, become localized to implantation sites during normal rodent gestation.

A study of morphological and haemodynamic determinants of testicular echotexture characteristics in the ram.

The ultrasonographic image of an organ is a product of scattering and reflection of high-frequency ultrasound beams by discrete units of tissue. The number of acoustic tissue interfaces and vascularity affects the quantitative characteristics of grey-scale ultrasonographic images. This study was undertaken to examine the influences of scrotal/testicular integument and blood flow on testicular echotexture parameters in the ram. Serial ultrasonographic images were obtained during surgical castration of 7 Rideau Arcott rams aged 20–22 weeks. The first 2 sets of images were taken through the scrotum, prior to and after induction of anaesthesia. The third set was taken through the tunica vaginalis, the fourth set was obtained through the tunica albuginea, the fifth set was taken when the testicular cord and internal blood vessels were clamped, and the final set of images was recorded after allowing the blood to drain from dissected testicles (5 min). All images were then subjected to computerized image analyses and the testicles were processed for histology. The removal of the scrotal skin and tunica vaginalis both resulted in significant (P < 0.05) increments in numerical pixel values (NPVs) and pixel heterogeneity (standard deviation of pixel values) of the testicular parenchyma. There were no differences (P > 0.05) in testicular echotexture between images taken just before or after clamping the testicular cord vessels, or after draining. At all stages, NPVs were correlated (P ≤ 0.10) to the seminiferous tubule (ST) area and the ST lumen area, except for NPVs and the ST lumen area in images obtained through the tunica albuginea (P = 0.20). We concluded that: 1) attenuation of ultrasound waves by the scrotal skin and tunica vaginalis significantly altered testicular echotexture characteristics; 2) vascular blood flow did not affect the echotextural attributes of the rams’ testes; and 3) NPVs were a good indicator of ST microstructure in situ and ex vivo.

Effects of disruption of the embryonic alkaline phosphatase gene on preimplantation development of the mouse

Embryonic alkaline phosphatase (EAP) is expressed during the preimplantation period of mouse development; however, its function is unknown. To determine whether the absence of an EAP gene affects development of preimplantation embryos, we studied mice homozygous for the disrupted EAP gene (EAP.ko mice). Time to reach morphologically definedpreimplantation stages, preimplantation loss, cell count, gestation length, and litter size were monitored, and it was found that EAP.ko embryos have slower development and higher rates of degeneration during in vitro preimplantation development. In vivo, EAP.ko mice had a longergestation, smaller litter size, and fewer cells at 93 hr after human chorionic gonadotropin injection. Furthermore, there was no compensation for the absence of EAP gene in EAP.ko embryos by other isozymes of alkaline phosphatase. We conclude that the presence of an active EAP gene is beneficial for preimplantation development of the mouse embryo, and its absence leads to fewer blastocysts in vitro, delayed parturition, and reduced litter size in vivo. Dev Den;217:440–448.

Development of strips of ovine testes after xenografting under the skin of mice and co-transplantation of exogenous spermatogonia with grafts

Xenografting of testicular tissue is an attractive new strategy for studying postnatal development of spermatogenesis and to preserve male genetics in large mammals. Typically, small cubes of immature testis (1 mm(3)) are grafted under the dorsal skin of immune-deficient mice. We attempted to increase the total number of seminiferous tubules in each xenograft with spermatogenesis by grafting flat strips of testis (approximately 9 x 5 x 1 mm) from ram lambs in immune-deficient mice. The percentage of grafts that survived and percentage of seminiferous tubules that developed spermatogenesis were the same as those reported after xenografting small cubes of lamb testis. Partially purified sheep spermatogonia were labeled with the fluorescent dye carboxy fluorescein diacetate succinyl diester and transplanted into the seminiferous tubules of one of the donor testis just before engraftment. The temporary label in the donor cells was detected for 4 weeks after xenografting, suggesting that co-engraftment of spermatogonia with testicular tissue may be a way to rapidly determine the effect of a specific gene on spermatogenesis. Finally, Sertoli cell lesions in xenografts of lamb testes were quantified, and their number and severity were found to increase, especially after grafts had been in place for 4 weeks. Although this coincided with the development of spermatogenesis, the extent of germ cell differentiation negatively correlated with severity of the lesions.

Sequences and expression patterns of alkaline phosphatase isozymes in preattachment bovine embryos and the adult bovine

We report the cloning and partial sequences of two novel bovine tissue‐specific alkaline phosphatase (AP) isozymes (TSAP2 and TSAP3) from in vitro–produced bovine blastocysts. Using a reverse‐transcribed polymerase chain reaction (RT‐PCR)–based assay for mRNA expression and in vitro–produced preattachment bovine embryos, TSAP2 mRNA was detected first at the four‐cell stage prior to the major burst of embryonic transcription in cattle and TSAP3 at the eight‐cell stage with the major burst in transcription. Furthermore, the transcription of TSAP2 and TSAP3 displays a curious “on‐off” pattern during early cleavages between 40 and 120 hr after insemination. Activity of bovine AP, measured by an azo‐dye coupling technique, indicates that at least one AP isozyme is functional in oocytes and embryos throughout bovine preattachment development. However, maternal and embryonic‐derived AP activity may have different cell‐surface distributions. This novel expression pattern of the bovine AP isozymes could provide a useful tool for identifying and clarifying the events controlling transcription and gene expression during early embryo development. Mol. Reprod. Dev. 50:7–17, 1998.

Suitability of epididymal and testicular ultrasonography and computerized image analysis for assessment of current and future semen quality in the ram.

Breeding soundness evaluation (BSE) is the primary assessment for determining the reproductive potential of male animals. This method, however, cannot be used to evaluate semen frequently or to predict future semen quality. Computerized analysis of ultrasonographic images provides information on histophysiological changes in male reproductive organs. We hypothesized that: (i) semen parameters would correlate with ultrasonographic characteristics of the distal region (cauda) of the epididymis and (ii) testicular ultrasound images and/or circulating testosterone concentration would predict future semen quality in the ram. Six adult rams underwent BSE and scrotal ultrasonography approximately 60 d apart (average duration of the spermatogenic cycle) both during the breeding (December and February) and non-breeding (June and August) seasons. An inverse correlation was found between pixel intensity (numerical pixel values) of the epididymes and percentage of sperm in semen with normal morphology (r = −0.46, P < 0.05). Pixel heterogeneity (standard deviation of pixel values) correlated negatively with percentage of sperm with normal morphology (r = −0.42, P < 0.05) and directly with percentage of spermatozoa with abnormal tails (r = 0.43, P < 0.05). Pixel heterogeneity of testicular parenchyma obtained approximately 60 d prior to semen evaluation inversely correlated with percentage of sperm with normal morphology (r = −0.73, P < 0.01) and sperm progressive motility (r = −0.76, P < 0.01), and directly with percentage of sperm with abnormal tails (r = 0.72, P < 0.01) and loose heads (r = 0.79, P < 0.01). We concluded that scrotal ultrasonography combined with computer-assisted analyses of epididymal and testicular echotexture in the ram was a valuable method for determining certain current and future semen parameters, respectively.

Relationships of serum thyroid hormones and follicle-stimulating hormone concentrations to Sertoli cell differentiation during the first wave of spermatogenesis in euthyroid ram lambs

The main purpose of this study was to determine if temporal relationships exist between serum concentrations of free fractions of thyroxin (fT4) and triiodothyronine (fT3), follicle-stimulating hormone (FSH) levels, and Sertoli cell differentiation in euthyroid ram lamb testes. Additionally, testicular thyroid hormone (TH) receptors (TRs) were identified using immunohistochemistry and Western blot analysis. Weekly testicular biopsies and jugular blood samples were collected from 12 ram lambs over the 9 weeks of study. Hormone concentrations and the numbers of dividing Sertoli cells per seminiferous tubule (ST) area were analyzed relative to chronological age of animals and the two distinctive stages of Sertoli cell differentiation: (a) tight junction/ST lumen formation and (b) the onset of support mechanisms for the development of multiple germ cell types (presence of primary spermatocytes in >95% STs). Circulating FSH concentrations increased (p<0.05) immediately after first detection of ST lumen and reached a nadir (p<0.05) just prior to the end of the first wave of spermatogenesis. A decline in both fT4 and fT3 levels (p<0.05) occurred after Sertoli cells had formed the ST lumen and began supporting germ cell differentiation. There was a positive correlation between the numbers of proliferating Sertoli cells and serum fT4 (r=0.51, p<0.001) and fT3 (r=0.52, p<0.001) concentrations. TRs were expressed throughout the study period; however, prior to the formation of ST lumen, two isoforms were detected while only one TR isoform was present by the end of the first wave of spermatogenesis. Overall, the exit of Sertoli cells from the cell cycle that presages their final differentiation begins when THs and FSH levels are high, suggesting a permissive role of these hormones in the maturation of STs in prepubertal ram lambs.

Relationships of ultrasonographic and magnetic resonance image attributes to the histomorphology of ram testes

Declining male fertility has prompted investigations into the diagnostic methods that would permit frequent, non-invasive and accurate detection of changes in testicular histomorphology and the reproductive status of individuals. Ultrasonographic (U/S) and magnetic resonance (MR) imaging both have the potential to be used in this manner as associations have previously been described between the U/S and MR image attributes and histopathological changes in testicular tissue. The present study set out to determine if correlations exist between quantitative U/S and MR image attributes and histomorphological characteristics (total and luminal seminiferous tubule, ST area, and parenchymal cell density) of the excised ram testes, and to compare relative sensitivities of the imaging techniques. The echotextural/MR (input variables) and histological parameters (output variables) were analyzed by the Pearsons product moment correlations. Significant correlations were found for all imaging modalities, with the strongest overall correlation recorded for the T2 FAST SPIN ECHO (T2FSE) MR series (between mean numerical pixel values (NPVs) and total ST area; r=-0.93, p<0.001). The greatest number of significant correlations among quantitative image characteristics and histological attributes of testicular tissue were found for the 3 PLANE LOCALIZER (3 PLANE LOC) MR series, followed by the T2FSE MR, 3D FAST-SPOILED GRADIENT ECHO (3D FSPGRE) MR, U/S (7.5 MHz) imaging, and finally T1 SPIN ECHO (T1SE) MR series. No significant correlations were recorded between the quantitative attributes of T1SE images and ST lumen area or parenchymal cell density, or between the attributes of the 3D FSPGRE images and cell density. We concluded that there existed a potential practical application for both U/S and MR image techniques, combined with computer-assisted image analysis, to monitor the changes in testicular histomorphology and male reproductive health and fertility. Scrotal U/S remains a first-line imaging technique for the assessment of male reproductive health due mainly to its versatility and lower cost.