Potential innate immunity-related markers of endometrial receptivity and recurrent implantation failure (RIF).

The successful implantation of the embryo into a receptive endometrium is essential for the establishment of a viable pregnancy while recurrent implantation failure (RIF) is a real challenge in assisted reproduction. The maternal innate immune system, specifically the Toll-like receptors (TLRs), are involved in maintaining immunity in the female reproductive tract (FRT) required for fertility. In this study, we aimed to investigate the importance of innate immunity-related gene expression in the regulation of human fertility and as a prediction of potential outcome of in vitro fertilization - embryo transfer (IVF-ET), thus, we assessed the gene expression levels of TLR signalling molecules using quantitative real-time PCR between endometrial biopsies of healthy fertile women, and the patients experiencing RIF. Interestingly, our results showed that, TRIB2 and TLR9 genes were differentially expressed between the endometrial biopsies of healthy women and those with RIF. However, comparing expression levels of same genes between pre-receptive and receptive healthy endometrial biopsies showed different genes (ICAM1, NFKBIA, VCAM1, LIF, VEGFB, TLR5) had significantly altered expression, suggesting their involvement in endometrial receptivity. Thus, further investigations will enable us to better understand the role of these genes in the biology of FRT and as a possible target for the improvement of infertility treatments and/or development of non-hormonal contraception.

A new strain of Taylorella asinigenitalis shows differing pathogenicity in mares and Jenny donkeys.

BACKGROUND: Three horse mares inadvertently inseminated with semen from a Tayorella asinigenitalis-positive Jack donkey developed severe, purulent endometritis whereas two Jenny donkeys mated naturally to the same Jack donkey did not develop clinical signs of infection. OBJECTIVES: To isolate and identify the causative agent. STUDY DESIGN: Case report. METHODS: Endometrial swabs from the infected mares were cultured on selective and non-selective media under aerobic and microaerophilic conditions. Isolates were subjected to Gram staining, oxidase and catalase tests, the Monotayl Latex Agglutination test and PCR to test for both T. equigenitalis and T. asinigenitalis. In vitro antimicrobial susceptibility testing was performed and the bacterial isolate was genotyped using MLST. RESULTS: A new sequence type of T. asinigenitalis was confirmed. MAIN LIMITATIONS: A limited numbers of mares and donkeys are described. CONCLUSIONS: This strain of T. asinigenitalis causes a severe venereal infection in mares but not in Jenny donkeys.

Hormonal Regulation of Glucocorticoid Inactivation and Reactivation in αT3-1 and LßT2 Gonadotroph Cells.

The regulation of reproductive function by glucocorticoids occurs at all levels of the hypothalamo-pituitary-gonadal axis. Within the pituitary, glucocorticoids have been shown to directly alter gene expression in gonadotrophs, indicating that these cell types are sensitive to regulation by the glucocorticoid receptor. Whilst the major glucocorticoid metabolising enzymes, 11ß-hydroxysteroid dehydrogenase (11ßHSD; HSD11B1 and HSD11B2), have been described in human pituitary adenomas, the activity of these enzymes within different pituitary cell types has not been reported. Radiometric conversion assays were performed in αT3-1, LßT2 (gonadotrophs), AtT-20 (corticotrophs) and GH3 (somatolactotrophs) anterior pituitary cell lines, using tritiated cortisol, corticosterone, cortisone or 11-dehydrocorticosterone as substrates. The net oxidation of cortisol/corticosterone and net reduction of cortisone/11-dehydrocorticosterone were significantly higher in the two gonadotroph cells lines compared with the AtT-20 and GH3 cells after 4 h. Whilst these enzyme activities remained the same in αT3-1 and LßT2 cells over a 24 h period, there was a significant increase in glucocorticoid metabolism in both AtT-20 and GH3 cells over this same period, suggesting cell-type specific activity of the 11ßHSD enzyme(s). Stimulation of both gonadotroph cell lines with either 100 nM GnRH or PACAP (known physiological regulators of gonadotrophs) resulted in significantly increased 11ß-dehydrogenase (11ßDH) and 11-ketosteroid reductase (11KSR) activities, over both 4 and 24 h. These data reveal that gonadotroph 11ßHSD enzyme activity can act to regulate local glucocorticoid availability to mediate the influence of the HPA axis on gonadotroph function.

Efficient isolation, biophysical characterisation and molecular composition of extracellular vesicles secreted by primary and immortalised cells of reproductive origin.

Effective communication between the maternal reproductive tract, gametes and the pre-implantation embryo is essential for the successful establishment of pregnancy. Recent studies have recognised extracellular vesicles (EVs) as potent vehicles for intercellular communication, potentially via their transport of microRNAs (miRNAs). The aim of the current investigation was to determine the size, concentration and electrical surface properties (zeta potential) of EVs secreted by; (1) primary cultures of porcine oviductal epithelial cells (POECs) from the isthmus and ampullary regions of the female reproductive tract; (2) Ishikawa and RL95-2 human endometrial epithelial cell line cultures; and (3) the non-reproductive epithelial cell line HEK293T. In addition, this study investigated whether EVs secreted by POECs contained miRNAs. All cell types were cultured in EV-depleted medium for 24 or 48 h. EVs were successfully isolated from conditioned culture media using size exclusion chromatography. Nanoparticle tracking analysis (NTA) was performed to evaluate EV size, concentration and zeta potential. QRT-PCR was performed to quantify the expression of candidate miRNAs (miR-103, let-7a, miR-19a, miR-203, miR-126, miR-19b, RNU44, miR-92, miR-196a, miR-326 and miR-23a). NTA confirmed the presence of EVs with diameters of 50-150 nm in all cell types. EV size distribution was significantly different between cell types after 24 and 48 h of cell culture and the concentration of EVs secreted by POECs and Ishikawa cells was also time dependent. The distribution of EVs with specific electrokinetic potential measurements varied between cell types, indicating that EVs of differing cellular origin have varied membrane components. In addition, EVs secreted by POECs exhibited significantly different time dependant changes in zeta potential. QRT-PCR confirmed the presence of miR-103, let-7a, miR-19a, miR-203, miR-126, and miR-19b in EVs secreted by POECs (CT ≥ 29). Bioinformatics analysis suggests that these miRNAs are involved in cell proliferation, innate immune responses, apoptosis and cellular migration. In conclusion, reproductive epithelial cells secrete distinct populations of EVs containing miRNAs, which potentially act in intercellular communication in order to modulate the periconception events leading to successful establishment of pregnancy.

Alteration in the expression of the renin-angiotensin system in the myocardium of mice conceived by in vitro fertilization.

Epidemiological studies have revealed that offspring conceived by in vitro fertilization (IVF) have an elevated risk of cardiovascular malformations at birth, and are more predisposed to cardiovascular diseases. The renin-angiotensin system (RAS) plays an essential role in both the pathogenesis of congenital heart disease in fetuses and cardiovascular dysfunction in adults. This study aimed to assess the relative expression levels of genes in the RAS pathway in mice conceived using IVF, compared to natural mating with superovulation. Results demonstrated that expression of the angiotensin II receptor type 1 (AGTR1), connective tissue growth factor (CTGF), and collagen 3 (COL3), in the myocardial tissue of IVF-conceived mice, was elevated at 3 weeks, 10 weeks, and 1.5 years of age, when compared to their non-IVF counterparts. These data were supported by microRNA microarray analysis of the myocardial tissue of aged IVF-conceived mice, where miR-100, miR-297, and miR-758, which interact with COL3, AGTR1, and COL1 respectively, were upregulated when compared to naturally mated mice of the same age. Interestingly, bisulfite sequencing data indicated that IVF-conceived mice exhibited decreased methylation of CpG sites in Col1. In support of our in vivo investigations, miR-297 overexpression was shown to upregulate AGTR1 and CTGF, and increased cell proliferation in cultured H9c2 cardiomyocytes. These findings indicate that the altered expression of RAS in myocardial tissue might contribute to cardiovascular malformation and/or dysfunction in IVF-conceived offspring. Furthermore, these cardiovascular abnormalities might be the result of altered DNA methylation and abnormal regulation of microRNAs.

Minimum Effective Dose of Cattle and Sheep BSE for Oral Sheep Infection.

The minimum dose required to cause infection of Romney and Suffolk sheep of the ARQ/ARQ or ARQ/ARR prion protein gene genotypes following oral inoculation with Romney or Suffolk a sheep Bovine spongiform encephalopathy (BSE)-derived or cattle BSE-derived agent was investigated using doses ranging from 0.0005g to 5g. ARQ/ARQ sheep which were methionine (M) / threonine (T) heterozygous or T/T homozygous at codon 112 of the Prnp gene, dosed ARQ/ARR sheep and undosed controls did not show any evidence of infection. Within groups of susceptible sheep, the minimum effective oral dose of BSE was found to be 0.05g, with higher attack rates following inoculation with the 5g dose. Surprisingly, this study found no effect of dose on survival time suggesting a possible lack of homogeneity within the inoculum. All clinical BSE cases showed PrPd accumulation in brain; however, following cattle BSE inoculation, LRS involvement within Romney recipients was found to be significantly lower than within the Suffolk sheep inoculated group which is in agreement with previous reports.

Objects in Contact with Classical Scrapie Sheep Act as a Reservoir for Scrapie Transmission.

Classical scrapie is an environmentally transmissible prion disease of sheep and goats. Prions can persist and remain potentially infectious in the environment for many years and thus pose a risk of infecting animals after re-stocking. In vitro studies using serial protein misfolding cyclic amplification (sPMCA) have suggested that objects on a scrapie-affected sheep farm could contribute to disease transmission. This in vivo study aimed to determine the role of field furniture (water troughs, feeding troughs, fencing, and other objects that sheep may rub against) used by a scrapie-infected sheep flock as a vector for disease transmission to scrapie-free lambs with the prion protein genotype VRQ/VRQ, which is associated with high susceptibility to classical scrapie. When the field furniture was placed in clean accommodation, sheep became infected when exposed to either a water trough (four out of five) or to objects used for rubbing (four out of seven). This field furniture had been used by the scrapie-infected flock 8 weeks earlier and had previously been shown to harbor scrapie prions by sPMCA. Sheep also became infected (20 out of 23) through exposure to contaminated field furniture placed within pasture not used by scrapie-infected sheep for 40 months, even though swabs from this furniture tested negative by PMCA. This infection rate decreased (1 out of 12) on the same paddock after replacement with clean field furniture. Twelve grazing sheep exposed to field furniture not in contact with scrapie-infected sheep for 18 months remained scrapie free. The findings of this study highlight the role of field furniture used by scrapie-infected sheep to act as a reservoir for disease re-introduction although infectivity declines considerably if the field furniture has not been in contact with scrapie-infected sheep for several months. PMCA may not be as sensitive as VRQ/VRQ sheep to test for environmental contamination.

Dynamics of the natural transmission of bovine spongiform encephalopathy within an intensively managed sheep flock.

Sheep are susceptible to the bovine spongiform encephalopathy (BSE) agent and in the UK they may have been exposed to BSE via contaminated meat and bone meal. An experimental sheep flock was established to determine whether ovine BSE could be naturally transmitted under conditions of intensive husbandry. The flock consisted of 113 sheep of different breeds and susceptible PRNP genotypes orally dosed with BSE, 159 sheep subsequently born to them and 125 unchallenged sentinel controls. BSE was confirmed in 104 (92%) orally dosed sheep and natural transmission was recorded for 14 of 79 (18%) lambs born to BSE infected dams, with rates varying according to PRNP genotype. The likelihood of natural BSE transmission was linked to stage of incubation period of the dam: the attack rate for lambs born within 100 days of the death of BSE infected dams was significantly higher (9/22, 41%) than for the rest (5/57, 9%). Within the group of ewes lambing close to death, those rearing infected progeny (n = 8, for 9/12 infected lambs) showed a significantly greater involvement of lymphoid tissues than those rearing non-infected offspring (n = 8, for 0/10 infected lambs). Horizontal transmission to the progeny of non-infected mothers was recorded only once (1/205, 0.5%). This low rate of lateral transmission was attributed, at least partly, to an almost complete absence of infected placentas. We conclude that, although BSE can be naturally transmitted through dam-lamb close contact, the infection in this study flock would not have persisted due to low-efficiency maternal and lateral transmissions.

Experimental oral transmission of atypical scrapie to sheep.

To investigate the possibility of oral transmission of atypical scrapie in sheep and determine the distribution of infectivity in the animals' peripheral tissues, we challenged neonatal lambs orally with atypical scrapie; they were then killed at 12 or 24 months. Screening test results were negative for disease-specific prion protein in all but 2 recipients; they had positive results for examination of brain, but negative for peripheral tissues. Infectivity of brain, distal ileum, and spleen from all animals was assessed in mouse bioassays; positive results were obtained from tissues that had negative results on screening. These findings demonstrate that atypical scrapie can be transmitted orally and indicate that it has the potential for natural transmission and iatrogenic spread through animal feed. Detection of infectivity in tissues negative by current surveillance methods indicates that diagnostic sensitivity is suboptimal for atypical scrapie, and potentially infectious material may be able to pass into the human food chain.

The natural atypical scrapie phenotype is preserved on experimental transmission and sub-passage in PRNP homologous sheep.

BACKGROUND: Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease.However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007. RESULTS: This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype. CONCLUSIONS: Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.

Expression and activities of 11betaHSD enzymes in the testes and reproductive tracts of sexually immature male pigs.

In light of studies implicating glucocorticoids in the control of testicular steroidogenesis and/or spermatogenesis, the objective of this study was to characterise the expression and activities of the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes in the testis and reproductive tract of the pre-pubertal pig. Although 11betaHSD1 and 11betaHSD2 mRNA transcripts and proteins were co-expressed in all regions of the reproductive tract, cortisol-cortisone inter-conversion was detectable in the testis, caput epididymidis and bulbourethral glands only. In homogenates of these 3 tissues, the apparent K(m) for NADP(+)- and NAD(+)-dependent 11beta-dehydrogenase activities ranged between 152-883 and 47-479 nmoll(-1), respectively. Irrespective of the pyridine nucleotide co-substrate, estimates of V(max) were consistently two orders of magnitude higher in the testis. Moreover, while, in each tissue, levels of cortisol oxidation were comparable in the presence of either NADP(+) or NAD(+), maximal rates of NAD(P)(+)-dependent cortisol oxidation were up to 33-fold greater than the V(max) for NADPH-dependent reduction of cortisone. We conclude that in the testis, caput epididymidis and bulbourethral gland of the immature pig, NADP(+)- and NAD(+)-dependent 11betaHSD enzymes catalyse net inactivation of cortisol, suggesting a physiological role for these enzymes in limiting local actions of glucocorticoids within these male reproductive tissues prior to puberty.

11Beta-hydroxysteroid dehydrogenase enzymes in the testis and male reproductive tract of the boar (Sus scrofa domestica) indicate local roles for glucocorticoids in male reproductive physiology.

11Beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes modulate the target cell actions of corticosteroids by catalysing metabolism of the physiological glucocorticoid (GC), cortisol, to inert cortisone. Recent studies have implicated GCs in boar sperm apoptosis. Hence, the objective of this study was to characterise 11betaHSD enzyme expression and activities in the boar testis and reproductive tract. Although 11betaHSD1 and 11betaHSD2 mRNA transcripts and proteins were co-expressed in all tissues, cortisol-cortisone interconversion was undetectable in the corpus and cauda epididymides, vas deferens, vesicular and prostate glands, irrespective of nucleotide cofactors. In contrast, homogenates of boar testis, caput epididymidis and bulbourethral gland all displayed pronounced 11betaHSD activities in the presence of NADPH/NADP(+) and NAD(+), and the penile urethra exhibited NAD(+)-dependent 11beta-dehydrogenase activity. In kinetic studies, homogenates of boar testis, caput epididymidis and bulbourethral gland oxidised cortisol with K(m) values of 237-443 and 154-226 nmol/l in the presence of NADP(+) and NAD(+) respectively. Maximal rates of NADP(+)-dependent cortisol oxidation were 7.4- to 28.5-fold greater than the V(max) for NADPH- dependent reduction of cortisone, but were comparable with the rates of NAD(+)-dependent cortisol metabolism. The relatively low K(m) estimates for NADP(+) -dependent cortisol oxidation suggest that either the affinity of 11betaHSD1 has been increased or the cortisol inactivation is catalysed by a novel NADP(+)-dependent 11betaHSD enzyme in these tissues. We conclude that in the boar testis, caput epididymidis and bulbourethral gland, NADP(+)- and NAD(+)-dependent 11betaHSD enzymes catalyse net inactivation of cortisol, consistent with a physiological role in limiting any local actions of GCs within these reproductive tissues.

Implication of cortisol and 11beta-hydroxysteroid dehydrogenase enzymes in the development of porcine (Sus scrofa domestica) ovarian follicles and cysts.

This study investigated cortisol inactivation by 11beta-hydroxysteroid dehydrogenase (11beta HSD) enzymes in porcine granulosa cells from antral follicles at different developmental stages and in ovarian cysts. In granulosa cells, cortisol oxidation increased threefold with antral follicle diameter (P < 0.001). This trend was paralleled by a threefold increase in NADP(+)-dependent 11beta-dehydrogenase activity in granulosa cell homogenates with follicle diameter. Intact granulosa cells from ovarian cysts exhibited significantly lower enzyme activities than cells from large antral follicles. Neither intact cells norcell homogenates displayed net 11-ketosteroid reductase activities. Since porcine follicular fluid (FF) from large antral follicles and ovarian cysts contains hydrophobic inhibitors of glucocorticoid metabolism by type 1 11beta HSD, this studyalso investigated whether levels of 11beta HSD inhibitors changed during follicle growth and could affect cortisol metabolism in granulosa cells. The extent of inhibition of 11beta HSD1 activity in rat kidney homogenates decreased progressively from 50 +/- 8% inhibition by FF from small antral follicles (P < 0.001) to 23 +/- 6% by large antral FF (P < 0.05). Cyst fluid inhibited 11beta HSD1 activity by 59 +/- 4% (P < 0.001). Likewise, net cortisol oxidation in granulosa cells was significantly decreased by large antral FF (35-48% inhibition, P < 0.05) and cyst fluid (45-75% inhibition, P < 0.01). We conclude that inactivation of cortisol by 11beta HSD enzymes in porcine granulosa cells increases with follicle development but is significantly decreased in ovarian cysts. Moreover, changes in ovarian cortisol metabolism are accompanied by corresponding changes in the levels of paracrine inhibitors of 11beta HSD1 within growing ovarian follicles and cysts, implicating cortisol in follicle growth and cyst development.

Complementation of daptomycin dptA and dptD deletion mutations in trans and production of hybrid lipopeptide antibiotics.

Daptomycin is a lipopeptide antibiotic produced by Streptomyces roseosporus and recently commercialized as Cubicin (daptomycin-for-injection) for treatment of skin and skin-structure infections caused by Gram-positive pathogens. Daptomycin is synthesized by a non-ribosomal peptide synthetase (NRPS) encoded by three overlapping genes, dptA, dptBC and dptD. The dptE and dptF genes, immediately upstream of dptA, are likely to be involved in the initiation of daptomycin biosynthesis by coupling decanoic acid to the N-terminal Trp. Analysis of RT-PCR data suggests that dptE, dptF, dptA, dptBC, dptD and possibly other dpt genes are transcribed as one large message; however, it has been demonstrated that sequential translation of these genes from a long transcript is not essential for robust daptomycin production. The dptA and the dptD genes were deleted from the dpt gene cluster, and expressed from ectopic positions in the chromosome under the control of the strong constitutive ermEp* promoter to produce high levels of lipopeptides. This three-locus trans-complementation system was used to produce hybrid lipopeptide antibiotics by introducing the heterologous lptD and cdaPS3 genes from Streptomyces fradiae and Streptomyces coelicolor, respectively, to complement the DeltadptD mutation.

The significance of cooling rates and animal variability for boar sperm cryopreservation: insights from the cryomicroscope.

Once the first methods for freezing mammalian semen had been established, research aimed at improving cryopreservation procedures became highly focused on the interactions between cooling rates and the permeability of the plasma membrane to water and cryoprotectants. This was based on the premise that cooling rates could be optimized from a theoretical basis for different species of interest. While this approach has stimulated considerable research, it has not achieved its original aim at the species level, largely because it overlooks inter-individual variation in sperm biochemical composition and physiology. If the underlying hypothesis is valid, however, optimal cooling rates should be identifiable for spermatozoa from individual animals. Experiments with the cryomicroscope revealed that while sperm survival after cryopreservation varied considerably between boars, there was little evidence that optimal freezing rates could be identified for individuals. Based on these findings, we tested the hypothesis that sperm susceptibility to cryoinjury is a consistent feature of each individual, but those individuals differ in susceptibility. This hypothesis was supported by evidence from an experiment with >100 boars; moreover, using genetic analyses, we demonstrated genomic differences between individual boars that correlated with post-thaw sperm quality.

Glucocorticoid metabolism and reproduction: a tale of two enzymes.

Within potential target cells, the actions of physiological glucocorticoids (cortisol and corticosterone) are modulated by isoforms of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). To date, two isoforms of 11 beta HSD have been cloned: 11 beta HSD1 acts predominantly as an NADP(H)-dependent reductase to generate active cortisol or corticosterone, and 11 beta HSD2 is a high affinity NAD(+)-dependent enzyme that catalyses the enzymatic inactivation of glucocorticoids. Whereas the regeneration of active glucocorticoids by 11 beta HSD1 has been implicated in the cellular mechanisms of pituitary function, ovulation and parturition, the enzymatic inactivation of cortisol and corticosterone by 11 beta HSD enzymes appears to be central to the protection of gonadal steroidogenesis, prevention of intra-uterine growth retardation, and lactation. Recent evidence indicates that follicular fluid contains endogenous modulators of cortisol metabolism by 11 beta HSD1, the concentrations of which are associated with the clinical outcome of assisted conception cycles and are altered in cystic ovarian disease. In conclusion, the two cloned isoforms of 11 beta HSD fulfil diverse roles in a wide range of reproductive processes from conception to lactation.

Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison.

This study compared variation in the quality of cryopreserved boar spermatozoa and the control and accuracy of cooling rates between three semen freezers (CryoLogic Freeze Control CL3000, Planer Products Kryo Save Compact KS1.7/Kryo 10 Control module and a controlled rate 'Watson' freezing machine developed within our laboratory). Five ejaculates were collected from each of 15 boars (five boars from each of three breeds). Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% v/v glycerol) and placed into 0.5 ml straws. Three straws per treatment, from each ejaculate were cooled to -5 degrees C at 6 degrees C/min, held at -5 degrees C for 30s while ice crystal formation was induced, then further cooled from -5 to 80 degrees C at either 40 degrees C/min (Kryo Save Compact KS1.7 and Watson) or 6 degrees C/min (Freeze Control CL3000). Precise measurements of temperature fluctuations during the programmed cooling curves were made by inserting thermocouples into the semen filled straws. Semen was assessed for %motile cells, motility characteristics using computer-assisted semen analysis (CASA), plasma membrane integrity (%SYBR-14 positive stained spermatozoa) and acrosome integrity (%FITC-PNA positive stained spermatozoa). Spermatozoa cryopreserved using the Freeze Control CL3000 system (maximum rate of 6 degrees C/min) exhibited reduced post-thaw viability (14.2+/-2.8% mean plasma membrane intact spermatozoa) when compared to both the KS1.7 and Watson freezers (optimal rate of 40 degrees C/min) (18.4+/-3.2 and 25.7+/-3.7% mean plasma membrane intact spermatozoa, respectively). Differences in motility characteristics were observed between spermatozoa cryopreserved at 40 degrees C/min with the Watson apparatus preserving a larger proportion of sperm with progressive motility. Cooling curves in the CL3000 and KS1.7 were interrupted by a pronounced increase in temperature at -5 degrees C that corresponded with the latent heat of fusion released with ice crystal formation. This temperature change was significantly reduced in the cooling curves produced by the Watson freezer. These findings suggest that preserving spermatozoa using the Watson freezer improved post-thaw semen quality, with regard to sperm motility characteristics. Furthermore, that post-thaw semen viability was enhanced by minimising temperature fluctuations resulting from the release of the latent heat of fusion at ice crystal formation.

Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11beta HSD) activity in follicular fluid from bovine and porcine large antral follicles and spontaneous ovarian cysts.

In the ovary, cortisol is oxidized to cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). The present study investigated whether follicular fluid (FF) from large antral follicles and spontaneous ovarian cysts, isolated from bovine and porcine ovaries, contained modulators of 11betaHSD activity. Whereas FF from antral follicles had no significant effect over 1 h on NADP+-dependent 11betaHSD activity in rat kidney homogenates, enzyme activity was inhibited by FF from bovine and porcine ovarian cysts (80.5% +/- 2.3% and 72.8% +/- 3.4% of control, respectively). Following C18 reverse-phase chromatography, the hydrophilic fractions of FF from bovine and porcine antral follicles stimulated NADP+-dependent 11betaHSD activities (111.5% +/- 21.6% and 55.2% +/- 5.7% respectively). Hydrophobic compounds inhibited NADP+-dependent cortisol oxidation by 58.2% +/- 5.1% (bovine) and 45.7% +/- 2.0% (porcine). In both species, FF from ovarian cysts appeared to contain less of the hydrophilic stimuli to 11betaHSD activity and more of the hydrophobic inhibitors. The FF from antral follicles and ovarian cysts, and the C18 fractions thereof, had no significant effect on NAD+-dependent cortisol oxidation. The ovarian modulators of NADP+-dependent 11betaHSD activities did not coelute with cortisol, cortisone, estradiol, testosterone, progesterone, pregnenolone, and cholesterol. However, the 11betaHSD stimuli in porcine FF from both antral follicles and cysts coeluted with prostaglandin (PG) E2 and PGF2alpha. We conclude that large antral follicles and spontaneous ovarian cysts, in both the cow and the pig, contain ovarian modulators of the NADP+-dependent 11betaHSD activity. Moreover, FF from spontaneous ovarian cysts, because of decreased content of the 11betaHSD stimulus accompanied by increased content of the 11betaHSD inhibitors, exerts a net inhibitory effect on 11betaHSD activity.