Short communication: Heat treatment and souring do not affect milk estrone and 17ß-estradiol concentrations.

The aim of our study was to establish whether heat treatment and souring of milk affect its estrone (E1) and 17ß-estradiol (E2) concentrations. Milk samples were collected from 10 Holstein cows in late pregnancy. Concentrations of E1 and E2 were measured in milk samples that were previously heated to 70 and 95°C for 5 min. Additionally, E1 and E2 concentrations were determined in the same milk samples after 2 d of spontaneous souring at room temperature, and these samples were compared with E1 and E2 levels in raw, unprocessed milk. Concentrations of both hormones were determined by commercial ELISA kits. Concentrations of E1 in unprocessed and processed milk (milk heated to 70 and 95°C and soured milk) were (mean ± SE) 47.25 ± 4.16, 44.84 ± 3.47, 41.00 ± 4.55, and 44.92 ± 3.91 pg/mL, respectively. Concentrations of E2 in the same milk samples were 36.11 ± 10.01, 32.46 ± 9.88, 31.78 ± 9.56, and 31.43 ± 8.00 pg/mL, respectively. Concentrations of E1 and E2 in heat-treated milk did not differ significantly from those in unprocessed milk. Similarly, E1 and E2 concentrations in soured milk did not differ significantly from those in unprocessed milk samples. These results indicate that E1 and E2 are stable in milk and that milk processing (heating and souring) does not influence their degradation. Therefore, E1 and E2 concentrations are expected to be similar between commercial full-fat milk and the raw milk from which it was produced.

Effect of dietary estrogens from bovine milk on blood hormone levels and reproductive organs in mice.

Cows are often milked until 60 d before their next expected calving. Milk from cows in the third trimester of pregnancy contains up to 20 times more estrogens than milk from nonpregnant cows. The aim of this study was to evaluate whether exposure to known doses of estrogens from bovine milk could affect blood hormone levels in mice and influence their reproductive organs. This study was performed with 30 intact male and 30 ovariectomized female mice. Mice of each sex were randomly divided into 3 experimental groups, each with 6 animals of each sex, and a control group with 12 animals of each sex. The first experimental group received 4mL of milk each day from a pregnant cow with natural estrone (E1) and 17ß-estradiol (E2) in concentrations 0.093 and 0.065ng/mL, respectively. The second experimental group received 4mL of the same milk each day, with an added 10ng/mL of both E1 and E2. The third experimental group received 4mL of the same milk each day, with an added 100ng/mL of both E1 and E2. The control group received no milk. After 8 d of treatment, mice were euthanized, blood was collected, and the uteruses, testes, and seminal vesicles were weighed. The results of our study demonstrated that consumption of native milk from a pregnant cow did not affect plasma E1 and E2 levels in either sex; uterine weight in females; or testosterone levels and testes and seminal vesicle weights in males. Similarly, we found no changes in the group that received the milk with an added 10ng/mL of E1 and E2. We did observe elevated plasma estrogens in both sexes, increased uterus weight in females, and decreased plasma testosterone levels in males from the group that received milk with an added 100ng/mL of E1 and E2. However, concentrations in the third group exceeded the physiological concentration of milk estrogens by 1,000 times, so it would be extremely unlikely to find such concentrations in native cow milk.

Chronic ingestion of Porphyromonas gingivalis induces systemic nitric oxide response in mice.

INTRODUCTION: Porphyromonas gingivalis induces nitric oxide (NO) production in various cells, systemic NO elevation being expected in chronic oral challenge. METHODS: Groups of BALB/c mice were inoculated orally with either live P. gingivalis ATCC 33277 or sterile broth on days 0, 2 and 4, with or without later administration of the inducible nitric oxide synthase (iNOS) inhibitor 1400W. Plasma and tissues were harvested on day 42 for assays of tumor necrosis factor-alpha (TNF-alpha), nitrite and nitrate (NOx) and tissue NO, or histology and iNOS immunohistochemistry. RESULTS: No signs of gingivitis were observed, but plasma NOx was significantly elevated (P = 0.028) as was TNF-alpha (P = 0.079) in P. gingivalis-inoculated animals compared with controls, NOx being reduced when 1400W was used. NO production in organs showed a similar trend, with significant elevation in liver (P = 0.017) and kidneys (P = 0.027), whereas concomitant treatment of inoculated animals with 1400W caused significant reductions in NO in aorta (P = 0.008) and kidneys (P = 0.046). Sham-inoculated 1400W-treated animals had significantly increased plasma NOx (P = 0.004) and liver NO (P = 0.04). NOx in plasma correlated significantly with NO production in lungs (0.35, P = 0.032) and kidneys (0.47, P = 0.003). Immunohistochemistry demonstrated iNOS activity in many tissues in all groups. CONCLUSION: Repeated oral administration of P. gingivalis induced systemic NO and NOx production in mice, probably by activating iNOS as suggested by the response to 1400W.

Identification of differentially expressed genes in 4-day axolotl limb blastema by suppression subtractive hybridization.

The goal of our study was the identification of up-regulated genes during axolotl (Ambystoma mexicanum) hindlimb regeneration 4 days after amputation using suppression subtractive hybridization (SSH). Approximately 400 clones that harbored upregulated genes in regenerating blastema tissue were selected for sequence analysis. A BLAST homology search against NCBI non-redundant database and an ambystoma EST database revealed 102 clones that showed homology to known sequences in GenBank with annotated function, 31 were known genes without known function, 74 were novel and 72 belonged to mitochondrial sequences. Differential expression of Hmox1, Orc4L, Pls3, Fen-1, Mcm7 and Mmp3/10a was confirmed using qRT-PCR analysis. Among all genes, only Mmp3/10a has been previously described as involved in limb regeneration. Other important identified genes belong to the group of cell cycle regulators (Orc4L, Nasp, Skp1A and Mcm7, the latter being a possible proliferative marker), those involved in protein synthesis and transport (Sec63, Srp72, Sara2) and V- ATPase pump. The novel genes we identified might be important for the process of blastema formation and the onset of cell proliferation in a regenerating limb.

Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice.

Steroidogenic acute regulatory protein (StAR) is essential for adrenal and gonadal steroidogenesis, stimulating the translocation of cholesterol to the inner mitochondrial membrane where steroidogenesis commences. StAR mutations in humans cause congenital lipoid adrenal hyperplasia (lipoid CAH), an autosomal recessive condition with severe deficiencies of all classes of steroid hormones. We previously described StAR knockout mice that mimic many features of lipoid CAH patients. By keeping StAR knockout mice alive with corticosteroid replacement, we now examine the temporal effects of StAR deficiency on the structure and function of steroidogenic tissues. The adrenal glands, affected most severely at birth, exhibited progressive increases in lipid deposits with aging. The testes of newborn StAR knockout mice contained scattered lipid deposits in the interstitial region, presumably in remnants of fetal Leydig cells. By 8 weeks of age, the interstitial lipid deposits worsened considerably and were associated with Leydig cell hyperplasia. Despite these changes, germ cells in the seminiferous tubules appeared intact histologically, suggesting that the StAR knockout mice retained some capacity for androgen biosynthesis. Sperm maturation was delayed, and the germ cells exhibited histological features of apoptosis, consistent with suboptimal androgen production. Immediately after birth, the ovaries of StAR knockout mice appeared normal. After the time of normal puberty, however, prominent lipid deposits accumulated in the interstitial region, accompanied by marked luteinization of stromal cells and incomplete follicular maturation that ultimately culminated in premature ovarian failure. These studies provide the first systematic evaluation of the developmental consequences of StAR deficiency in the various steroidogenic organs.

Freezability of equine semen after glass beads column separation.

REASONS FOR PERFORMING STUDY: The success rate of artificial insemination following the freezing of stallion semen is limited; therefore, improving the stallion semen quality after the freezing and thawing process is a necessary objective. OBJECTIVES: To investigate the influence of glass bead column separation on the freezability of stallion semen. HYPOTHESIS: Glass beads in a column separator remove damaged and dead spermatozoa in the ejaculate during centrifugation. METHODS: In total, 50 ejaculates from 6 Lipizzaner stallions were studied. Each ejaculate was divided into 2 parts, one half processed following standard procedure and the second half used for the column separation procedure. After freezing, semen quality was evaluated using standard tests for motility, morphology and viability of semen. RESULTS: Motility and progressive motility of the column-separated (CS) semen were significantly higher (P < 0.001) before freezing and immediately, 24 and 48 h after thawing. A significant increase (P < 0.001) in the percentage of hypoosmotic positive spermatozoa was observed in CS samples. The percentage of total morphological changes in the separated samples before and after freezing was significantly lower (P < 0.001) compared with samples prepared using the standard procedure. A substantial decrease (P < 0.001) was found in the percentage of spermatozoa with damaged acrosomes. However, the percentage of spermatozoa with coiled tails was increased in the separated samples (P < 0.001). CONCLUSIONS: Column separation before freezing has a positive effect on the quality of thawed equine semen. POTENTIAL RELEVANCE: The quality of CS frozen/thawed samples indicates their potential use for increasing insemination success in mares.

Differential patterns of expression of DAX-1 and steroidogenic factor-1 (SF-1) in the fetal rat testis.

Gonadal differentiation is dependent upon a cascade of molecular and morphological events. Steroidogenic factor 1 (SF-1) and DAX-1 have been implicated in this process. A SF-1 binding site has been reported to be present on the DAX-1 gene. We therefore used immunocytochemistry to determine whether these two transcription factors are co-expressed in the fetal testis. DAX-1 was immunolocalised to the cytoplasm of interstitial cells from fetal testis of rat and human from day 15.5 and 16 weeks of gestation respectively. SF-1 was detected in nuclei of fetal Sertoli and interstitial cells. In the fetal rat expression of SF-1 in interstitial cells was not uniform; cells with abundant SF-1 all contained 3-beta HSD and were classified as Leydig cells. DAX-1 expression was not exclusive to cells which contained SF-1 and SF-1/DAX-1 co-expressing cells were not exclusively fetal Leydig cells. We conclude that in the fetal testis expression of the DAX-1 protein is not dependent upon the presence of SF-1 although SF-1 is present at an earlier stage of gestation in the gonad. DAX-1 may therefore play a separate or complementary role to that of SF-1 in the modulation of testicular gene expression and differentiation.

Expression of cytochrome P450 17alpha-hydroxylase/C17-20 lyase in the fetal rat testis is reduced by maternal exposure to exogenous estrogens.

Testosterone is required for normal development of the male reproductive tract. Synthesis of testosterone occurs in the Leydig cells and is dependent upon the expression of several enzymes, including cytochrome P450 17alpha-hydroxylase/C17-20-lyase (P450c17), which is highly regulated within the testis. The aim of the present study was to investigate whether maternal exposure to estrogenic chemicals was able to affect Leydig cell function in the developing male fetus at the time of masculinization. Pregnant rats were injected sc with diethylstilbestrol (DES; 100 or 500 micrograms/kg), 4-octylphenol (OP; 100 or 600 mg/kg), or vehicle (oil, control) on days 11.5 and 15.5 postcoitum. Doses were chosen to reflect the reported estrogenic potency of the chemicals in vitro. On day 17.5, fetal testes were fixed before performing in situ hybridization and immunocytochemistry, used for extraction of RNA, or homogenized in phosphate buffer for determination of 17alpha-hydroxylase enzyme activity. There was no difference between fetuses from control and treated mothers in either the overall histology of the testes or the apparent number of Leydig cells, as determined by immunocytochemistry with an antibody directed against 3beta-hydroxysteroid dehydrogenase. However, there was a consistent and striking reduction in the amount of P450c17 detected by immunocytochemistry in testes from the groups given the higher dose of DES or OP. These observations were supported by measurement of 17alpha-hydroxylase activity, which was significantly reduced compared with that in controls (6.25 +/- 0.65 pmol/testis x min) in fetuses from animals treated with 100 micrograms/kg DES (4.27 +/- 0.39; P < 0.05), 500 micrograms/kg DES (1.4 +/- 0.47; P < 0.001), or 600 micrograms/kg OP (4.25 +/- 0.33; P < 0.05). RT-PCR and in situ hybridization revealed that these changes were mirrored by reductions in P450c17 messenger RNA in testes from fetuses from treated mothers compared with control levels. In conclusion, maternal treatment with either a potent sythetic estrogen (DES) or a putative environmental estrogen (OP) results in reduced expression of the messenger RNA and protein for P450c17 in fetal Leydig cells. These results, therefore, provide a mechanism by which inappropriate exposure of the fetus to estrogenic chemicals might have an adverse effect on fetal steroid synthesis and masculinization.

Testicular expression of inhibin and activin subunits and follistatin in the rat and human fetus and neonate and during postnatal development in the rat.

Inhibins, activins, and follistatins are all believed to play roles in the regulation of FSH secretion by the pituitary and in the paracrine regulation of testis function. Previous studies have resulted in conflicting data on the pattern of expression of the inhibin/activin subunits, and little information on expression of follistatin during fetal/neonatal life. We have made use of new, highly specific monoclonal antibodies and fixed tissue sections from fetal, neonatal, and adult rats, and limited amounts of fetal and neonatal human testis, to undertake a detailed immunocytochemical study of the pattern of expression of these regulatory proteins. In the rat, positive immunostaining for the alpha-subunit of inhibin (alpha) was first detectable on day 14.5 post coitum (p.c.), the first day on which the testis could be morphologically distinguished from the ovary. During fetal life, the alpha-immunostaining was most prominent in the fetal Leydig cells. In Sertoli cells, alpha-immunostaining was slightly stronger on days 14.5 and 15.5 p.c. compared with 16.5-20.5. After birth, alpha-immunostaining remained intense in fetal Leydig cells but declined following their replacement with their adult-type counterparts; in contrast, alpha-subunit increased in Sertoli cells immediately after birth. Immunostaining with antibodies specific to betaB-subunit showed a similar pattern to that of the alpha-subunit, except that positive immunostaining was first detectable on day 16.5 p.c., 2 days later than immunostaining for the alpha-subunit. The pattern of betaB-immunostaining in postnatal samples paralleled that of the alpha-subunit. Immunostaining using antibodies against the betaA-subunit did not produce any significant reaction product in any sample. Follistatin was undetectable in the fetal rat testis but appeared in the Leydig cells immediately after birth and its expression remained intense throughout postnatal development and in adult testis. No evidence was obtained for expression of either the inhibin/activin subunits or follistatin in the germ cells, peritubular myoid cells, or other interstitial cells in any of the sections examined. In the human fetal testis, both alpha- and betaB-subunits were immunodetectable at 16, 18, and 24 weeks gestation in Sertoli and Leydig cells, with stronger immunostaining in Sertoli cells at 24 weeks. Postnatally at 4 months, immunoexpression of the betaB-subunit was no longer detectable, whereas the alpha-immunostaining became weaker but was still present in both Sertoli and Leydig cells. No positive immunostaining for betaA-subunit or follistatin was detectable at any time point studied. In conclusion, we have shown that, in the rat testis, the majority of inhibin alpha-subunit and inhibin/activin betaB-subunit is immunolocalized to the fetal-type Leydig cells during fetal/neonatal life but, following birth, immunoexpression in the Sertoli cells of both subunits increases markedly while follistatin is immunodetectable only postnatally.

Immunolocalisation of androgen receptor to interstitial cells in fetal rat testes and to mesenchymal and epithelial cells of associated ducts.

Androgens are required for the development of male internal and external genitalia. Androgen action is mediated by an intracellular receptor which acts as a transcription factor following activation by ligand binding. The aim of the present study was to define the time of appearance of androgen receptor (AR) in the male fetal rat gonad using immunohistochemistry. Intact fetuses (days 13.5-16.5) or testicular tissue (days 16.5-20.5 and days 3-7 postnatal) were fixed in Bouins' solution and processed into paraffin wax. On day 16.5 nuclear AR were present in mesenchymal cells surrounding the Wolffian duct but those around the Mullerian duct were receptor negative. During the following day (17-18) the abundance of nuclear staining increased, becoming detectable in the epithelial cells of the Wolffian ducts. Within the testis some nuclear staining was apparent at day 17 but was confined to interstitial cells surrounding the seminiferous cords. As development of the testis proceeded the abundance of nuclear AR in peritubular and elongated mesenchymal cells increased. AR were not detected in fetal Leydig cells expressing 3 beta-hydroxysteroid dehydrogenase nor in the ovaries or associated ducts of female fetuses at the same ages. In conclusion, in the rat we have found AR expression detectable by immunohistochemistry in mesonephric mesenchyme to be confined to that underlying the Wolffian ducts and to be absent from the area around the degenerating Mullerian duct. On and after day 17 of gestation AR is present in Wolffian duct epithelial cell nuclei and within the testis it is confined to peritubular and interstitial cells which may have migrated from the mesonephros.(ABSTRACT TRUNCATED AT 250 WORDS)

Lanosterol 14alpha-demethylase (CYP51), NADPH-cytochrome P450 reductase and squalene synthase in spermatogenesis: late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterol.

Lanosterol 14alpha-demethylase (CYP51) is a cytochrome P450 enzyme involved primarily in cholesterol biosynthesis. CYP51 in the presence of NADPH-cytochrome P450 reductase converts lanosterol to follicular fluid meiosis activating sterol (FF-MAS), an intermediate of cholesterol biosynthesis which accumulates in gonads and has an additional function as oocyte meiosis-activating substance. This work shows for the first time that cholesterogenic enzymes are highly expressed only in distinct stages of spermatogenesis. CYP51, NADPH-P450 reductase (the electron transferring enzyme needed for CYP51 activity) and squalene synthase (an enzyme preceding CYP51 in the pathway) proteins have been studied. CYP51 was detected in step 3-19 spermatids, with large amounts in the cytoplasm/residual bodies of step 19 spermatids, where P450 reductase was also observed. Squalene synthase was immunodetected in step 2-15 spermatids of the rat, indicating that squalene synthase and CYP51 proteins are not equally expressed in same stages of spermatogenesis. Discordant expression of cholesterogenic genes may be a more general mechanism leading to transient accumulation of pathway intermediates in spermatogenesis. This study provides the first evidence that step 19 spermatids and residual bodies of the rat testis have the capacity to produce MAS sterols in situ.

Immunolocalisation of oestrogen receptor-alpha within the testis and excurrent ducts of the rat and marmoset monkey from perinatal life to adulthood.

The sites of action and the physiological role of oestrogens in the male reproductive tract are poorly understood. We have undertaken a systematic study of the immunoexpression of oestrogen receptor-alpha (ER alpha) in the male rat from late fetal life through to adulthood and compared the findings with results obtained in the marmoset monkey (Callithrix jacchus) from neonatal to adult life. The testes, rete testis, efferent ducts and epididymis were examined from normal male rats (aged 4, 8, 10, 15, 20, 25, 38, 48 and 90 days) and from male rat fetuses on days 17.5 and 18.5 of gestation; comparable tissues were examined from neonatal, infantile, peripubertal and adult marmosets aged 8, 18-24, 54-62 and 92-112 weeks respectively. Immunolocalisation of ER alpha used antigen retrieval and a monoclonal antibody directed to the N-terminus, which had proved superior to six other antisera tested. ER alpha was immunoexpressed in interstitial cells, including the fetal/ neonatal generation of Leydig cells, in both the rat and marmoset. In the rat, the adult generation of Leydig cells were also immunopositive for ER alpha whereas the comparable cells in the marmoset were only weakly immunopositive. ER alpha was not expressed in Sertoli cells, peritubular myoid cells, blood vessels or germ cells at any time in either species. In late fetal life in the rat, ER alpha was immunoexpressed in cells surrounding the mesonephric tubules, whereas postnatally it was expressed in the epithelium of the rete testis and efferent ducts at all ages from 4 to 90 days; this immunoexpression was most pronounced in the efferent ducts. In the marmoset, the efferent ducts, but not the rete testis, also showed intense immunoexpression of ER alpha. Apart from sporadic immunostaining for ER alpha in the epididymal duct of the rat in the neonatal period, the caput, corpus and cauda epididymis were negative for immunoexpression of ER alpha at all ages in both species. These findings suggest that the main actions of oestrogens in the male reproductive tract, mediated by ER alpha, are related to the development and function of the efferent ducts and the Leydig cells. In consideration of data from this and previous studies of oestrogen binding, we predict possible sites of expression of other oestrogen receptors (e.g. ER beta) in Sertoli cells and the epididymis. Interactive effects, related to the relative levels of androgens and oestrogens, could be physiologically important in the excurrent ducts of the adult testis.

Maternal oestrogen/xenoestrogen exposure alters expression of steroidogenic factor-1 (SF-1/Ad4BP) in the fetal rat testis.

Expression of steroidogenic factor 1 (SF-1/Ad4BP) is essential for gonadal differentiation. We have assessed whether expression of SF-1 in the gonads of rat fetuses was altered following maternal treatment with diethylstilbestrol (DES, a synthetic oestrogen) or 4-octylphenol (OP, a xenoestrogen). Pregnant rats were injected subcutaneously with DES (500 microg/kg), OP (600 mg/kg) or vehicle (oil, control) on days 11.5 and 15.5 post coitum (p.c.) and fetal rat testes were recovered on day 17.5 p.c. The level of expression of SF-1 was determined by RNase protection assay and immunocytochemistry. In both DES- and OP-exposed fetuses immunoexpression of SF-1 was reduced in Sertoli and interstitial cells when compared with controls. In parallel, a significant decrease occurred in the total amount of SF-1 mRNA (P < 0.05) in fetal testes but not in fetal ovaries. These results suggest that: (a) Sertoli cell-derived oestradiol may be important in the physiological regulation of SF-1 in the fetal testis; and (b) one mechanism by which inappropriate exposure to oestrogens might alter the genetic cascade that ensures normal development of the testis is via altered expression of SF-1.

Fetal and perinatal influence of xenoestrogens on testis gene expression.

The incidence of reproductive abnormalities in the male has been reported to have increased during the past 50 years. It has been suggested that these changes may be attributable to the presence of chemicals with oestrogenic activity in our environment. The aim of the experiments described in this chapter was to investigate the effects of acute exposure to high levels of xenoestrogens either indirectly during fetal life, or directly during neonatal life, on gene expression in the testis and pituitary. Fetal treatment involved administration of diethylstilbestrol (DES), 4-octylphenol (OP) or vehicle (oil, control) to pregnant rats on days 11.5 and 15.5 post coitum; fetuses were recovered on day 17.5. There was no difference between fetuses from control and treated mothers in either the overall histology of the testes or numbers of Leydig cells as determined by immunohistochemistry with an antibody directed against 3 beta-HSD. However there was a consistent and striking reduction in the amount of P450 17-a hydroxylase C17, 20 lyase (P450c17) and steroidogenic factor 1 (SF-1) detected by immunocytochemistry in testes from treatment groups given the higher doses of OP and DES. Oestrogen receptors (ER alpha) were present in the fetal leydig cells of all animals. Neonatal treatment involved direct injection of oil (control), DES, OP or Bisphenol A (Bis A) on days 2, 4, 6, 8, 10 and 12; pituitaries and testes were recovered on day 18. Testis weights and seminiferous tubule diameters were significantly reduced in animals treated with DES. In these same animals immunocytochemical localisation revealed that the amounts of FSH beta subunit and inhibin alpha subunit were reduced in their pituitaries and testes respectively. OP did not appear to have an acute, measurable effect on testis gene expression but a reduction in testis weight was noted in adult animals given the same treatment regime. The effects observed are consistent with negative feedback by oestrogens on pituitary production of FSH resulting in retarded maturation of seminiferous tubules and reduced Sertoli cell numbers. These studies have demonstrated that administration of high levels of oestrogens can affect gene expression in the testis early in life. However, the relevance of these findings to observations in man await a) a greater understanding of the physiological role(s) of oestrogens in normal males, b) an evaluation of the sources, routes of exposure, concentrations in vivo and bioavailability of xenoestrogens.

Combined histochemical and immunohistochemical determination of three muscle fibre types in a single section of porcine skeletal muscle.

The method of successive staining for SDH and immunohistochemical detection of fast contracting muscle fibres in pig muscle tissue has been used for demonstration of three muscle fibre types (slow oxidative, SO; fast metabolic intermediate, FI; fast glycolytic, FG). It gives basic information on contractile and metabolic muscle fibre properties in a single frozen section and can be well applied to the pale, soft and exudative (PSE) changes affecting pig muscles.

Effects of season, age, and breed on semen characteristics in different Bos taurus breeds in a 31-year retrospective study.

Besides genetic factors, the season of semen collection could have an important effect on ejaculate characteristics, although results from previously published studies are somewhat variable. To determine seasonal effects on semen characteristics, we have analyzed 71,983 ejaculates collected from bulls of four different breeds over a 31-year period. Ejaculate volume, semen concentration, and total sperm output were analyzed with the respect to season and age of bull. Results revealed that semen concentration did not vary significantly during seasons, and ejaculate volume and total sperm output are influenced by season in all breeds. The highest ejaculate volume and total number of sperm in ejaculates were observed during the summer, followed by spring, autumn, and winter. Results suggest that the gradual increase in the day length in the spring is most likely responsible for the highest sperm output during the summer months, suggesting that seasonal effects are also present in cattle, which is not normally considered a seasonal species.

Steroidogenic factor 1 and the central nervous system.

Steroidogenic factor 1 (SF-1; officially designated NR5a1) is a member of a nuclear receptor superfamily with important roles in the development of endocrine systems. Studies with global and tissue-specific (i.e. central nervous system) knockout mice have revealed several roles of SF-1 in brain. These include morphological effects on the development of the ventromedial nucleus of the hypothalamus and functional effects on body weight regulation through modulation of physical activity, anxiety-like behaviours and female sexual behaviours. Although such defects are almost certainly a result of the absence of SF-1 acting as a transcription factor in the hypothalamus, global SF-1 knockout mice also represent a model for studying the sex differences in the brain that develop in the absence of exposure to foetal sex steroid hormones as a result of the absence of gonads. In the present review, current knowledge of the roles of SF-1 protein in the central nervous system is discussed.

Initiation of steroidogenesis precedes expression of cholesterologenic enzymes in the fetal mouse testes.

Sexual differentiation is a carefully regulated process that ultimately results in a development of the male or female phenotype. Proper development of the male phenotype is dependent upon the action of testosterone and anti-mullerian hormone. Leydig cells start to produce testosterone around day 12.5 in the fetal mouse testis, and continue to produce high levels of this hormone throughout gestation. In the present study, we examined whether expression of lanosterol 14alpha-demethylase (cyp51) and cytochrome P450 NADPH reductase, both involved in the cholesterol production, occurs simultaneously with proteins required for the production of steroid hormones. Immunocytochemical staining with the antibodies against cyp51, cytochrome P450 NADPH reductase, steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase I (3beta-HSD I) was used to determine the ontogeny of expression of these four proteins. As expected, 3beta-HSD I and StAR proteins were detected on day 12.5 p.c., while expression of cyp51 and NADPH cytochrome P450 reductase appeared 1 day later, on day 13.5. Thereafter, the expression of all four proteins remained strong throughout gestation. Results of this study suggest that initial steps of steroid hormone production in murine Leydig cells are mostly dependent on exogenously derived cholesterol, while from day 13.5 onwards, mouse Leydig cells are able to synthesize cholesterol and are therefore not dependent on exogenous cholesterol resources.

Establishment of a pregnancy following intravaginal insemination with epididymal semen from a dog castrated due to benign prostatic hyperplasia.

Benign prostatic hyperplasia was diagnosed in an American Staffordshire Terrier of high breeding value presenting concurrent haematuria. Castration as a treatment was synchronized with the oestrus cycle of a bitch selected for insemination. After castration the cauda epididymis was flushed with Gent semen extender and collected spermatozoa were filtered and analysed by Hamilton Thorn computer assisted sperm analysis. A total of 7 ml semen containing 742 x 10(6) spermatozoa with 76.5% mean motility was used for insemination. Intravaginal insemination of the bitch was performed with an insemination catheter for dogs (Kruuse, Marslev, Denmark) on the day when plasma progesterone levels reached 9.9 ng/ml. Normal pregnancy without complications resulted in eight live-born puppies 63 days after insemination. This is the first report of a normal pregnancy and birth of puppies from a bitch inseminated with epididymal semen obtained from a dog affected by benign prostate hyperplasia.

Combinations of genetic changes in the human cAMP-responsive element modulator gene: a clue towards understanding some forms of male infertility?

The cAMP-responsive element modulator (CREM) gene plays a pivotal role in the mouse spermatogenesis, but its role in the human infertility has not been fully established. We performed a mutation screening in 13 Slovenian men with round spermatid arrest and in six controls. Eleven genetic changes have been identified in the human CREM gene, three novel single-nucleotide polymorphisms [within the promoters P1, P3 and intervening sequence 1 (IVS1)], one insertion (IVS2) and one non-sense mutation (exon gamma). Some infertile patients seem to accumulate potentially harmful genetic changes. We identified a patient with no CREM immunoreactive protein that was homozygous for the nucleotide changes in all promoters, IVS 1, 2, 6, and was heterozygous for the mutation in exon gamma. Interestingly, insertion in IVS2 (IVS2-58_55insT) results in a four-fold decrease in binding of nuclear proteins. Computer predictions suggested the presence of a potential novel CREM promoter, however, random amplification of cDNA ends from the human testis cDNA library was not successful in confirming a novel transcription start site of the CREM gene. Screening of a larger number of patients and controls is required to elucidate whether the observed combinations of genetic changes in the CREM gene can explain some forms of male infertility.