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.

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.

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.

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 suppressionsubtractive hybridization (SSH). Approximately 400 clones that harboredupregulated genes in regenerating blastema tissue were selected for sequence analysis.A BLAST homology search against NCBI non-redundant database and anambystoma EST database revealed 102 clones that showed homology to knownsequences in GenBank with annotated function, 31 were known genes withoutknown function, 74 were novel and 72 belonged to mitochondrial sequences. Differentialexpression of Hmox1, Orc4L, Pls3, Fen-1, Mcm7 and Mmp3/10a was confirmedusing qRT-PCR analysis. Among all genes, only Mmp3/10a has been previouslydescribed as involved in limb regeneration. Other important identified genesbelong to the group of cell cycle regulators (Orc4L, Nasp, Skp1A and Mcm7, the latterbeing a possible proliferative marker), those involved in protein synthesis andtransport (Sec63, Srp72, Sara2) and V- ATPase pump. The novel genes we identifiedmight be important for the process of blastema formation and the onset of cell proliferationin a regenerating limb (AU)

No disponible

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.

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.

Postnatal hypothyroidism does not affect prepubertal testis development in boars.

Young boars were treated with propiothiouracil to induce hypothyroidism to examine its effects on postnatal testicular development. Treatments with 0.1% 4-propyl-2-thiouracil (PTU) in drinking water started after weaning, at 3 weeks of age and all boars were severely hypothyroid at 6 weeks of age as determined by measuring T3 and T4 in blood plasma. Boars were castrated at different ages up to 20 weeks and their testes used for histological and immunohistochemical analyses. Although small but significant reduction in testis weight was observed from 8 to 12 weeks of age, this was not accompanied by significant difference in testicular volume. By 20 weeks of age, at the beginning of puberty, the differences in testis weights between control and treated groups of boars disappeared suggesting there is no lasting effect of hypothyroidism on postnatal development of boar testis. Immunohistochemical staining was used to determine the presence of molecular markers in both Sertoli and Leydig cells. Again, there were no differences between testes from control and treated boars in the pattern or intensity of immunostaining using antibodies against 3beta-hydroxysteroid dehydrogenase, antimullerian hormone or proliferating cell nuclear antigen (PCNA). Immunostaining with antibodies against PCNA showed interesting results as it was observed that Sertoli cells still express this marker of proliferating cells at 14 weeks of age, later than previously suggested cessation of Sertoli cell proliferation. This study suggests that hypothyroidism in boars does not have similar effects on postnatal testis development as reported in some other species.

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.

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.

Immunoexpression of the steroidogenic enzymes 3-beta hydroxysteroid dehydrogenase and 17 alpha-hydroxylase, C17,20 lyase and the receptor for luteinizing hormone (LH) in the fetal rat testis suggests that the onset of Leydig cell steroid production is independent of LH action.

The production of testosterone in the adult testis is mainly regulated by LH. Testosterone is essential for normal development of the male fetus. The regulation of testosterone production in the fetal testis is less clear than in the adult testis, and there are indications that at least the onset of androgen production in the fetal testis takes place in an LH-independent way. The aim of the present study was to compare the onset of synthesis and pattern of expression of LH receptors and two important steroidogenic enzymes, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17alpha-hydroxylase/17,20-lyase (P450c17), in fetal rat gonads. Whole fetuses (13.5 and 14.0 postcoitum (p.c.), testes, and ovaries dissected from fetuses on Days 14.5 p.c. to 20.5 p.c. and testes obtained on Days 3, 5, and 7 postpartum were fixed in Bouin's solution and processed for immunocytochemistry. In all samples of fetal testis, 3beta-HSD was detected on Day 14.5 p.c., and immunoexpression of P450c17 appeared one day later on Day 15.5 p.c. Thereafter, immunoexpression of both enzymes remained intense throughout gestation and postnatally. In contrast, immunoexpression of LH receptors was detectable only from Day 16.5, when a few weakly positive cells were present, but it became more intense one and two days later. Similar to the immunostaining for 3beta-HSD and P450c17, immunostaining for LH receptor remained strong throughout gestation and during the first 7 days of postnatal life. No immunoexpression of any of the three proteins studied was detected in the fetal ovary at any age. These data show very early immunoexpression of 3beta-HSD, which was detected one day before the reported start of steroid production by the fetal Leydig cells. In contrast, immunoexpression of LH receptors was evident only after immunoexpression of both the steroidogenic enzymes studied had became apparent and after the reported start of steroidogenesis, which is consistent with the theory that onset of steroidogenesis in the fetal rat testis is an LH-independent process.

Higher thyroid hormone levels in neonatal life result in reduced testis volume in postpubertal bulls.

Thyroid hormones appear to determine adult testis size in rodents by regulating the period of Sertoli cell proliferation in the neonatal period. In the present study, the correlation between neonatal thyroid hormone levels (T3 and thyroxin, T4) and postpubertal testis size in Simental bulls was examined. T3 and T4 levels were measured in blood plasma from 35 calves immediately after their arrival at the AI centre at age 3-6 months. Testis size (height and width) was measured at 12 months of age in the same live animals. A significant negative correlation (r = -0.55; p < or = 0.001) was found between T4 and calculated testicular volume using either the Pearson correlation test or linear regression analysis, while the levels of T3 and testis volume showed a negative correlation, although this did not reach statistical significance (r = -0.20, p < or = 0.05). The results of this study suggest, therefore, that neonatal thyroid hormone levels might have the same effect on testicular size in cattle as they do in rodents.

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.

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.

CYP17 gene analysis in hyperandrogenised women with and without exaggerated 17-hydroxyprogesterone response to ovarian stimulation.

Nine patients with polycystic ovary syndrome (PCOS) and exaggerated serum level of 17-hydroxyprogesterone (17-OHP) response to gonadotropin (GnRH) agonist stimulation, and seven patients with PCOS, but normal 17-OHP response have been analysed for possible linkage of PCOS with genetic defects on the 17 alpha-hydroxylase/17,20-lyase gene (CYP17). A portion of the regulatory and the entire coding domain for this enzyme have been analysed by PCR-SSCP analysis. Samples have been also screened for the previously reported -34 bp polymorphism, which creates a new SP1-type promoter site, but was excluded as the primary genetic defect. We have varied gel concentrations, reduced running temperatures, added glycerol to polyacrylamide gels and performed electrophoresis on longer gels in order to improve the resolving power of SSCP. Screening of the CYP17 gene revealed no mutations associated with the disease in the examined group of patients. Also, the -34 bp polymorphism proved to be equally distributed among patient and control samples, which in our case were non-related. The results indicate that, when germline mutations in question, CYP17 may be excluded as a candidate gene for these subtypes of PCOS.

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.