Semen quality and reproductive hormone levels in men from Southern Spain.

In North European countries, a significant difference in semen quality among young men has been shown. Men from the western countries, Denmark, Germany and Norway, have lower semen quality than men from the eastern countries Finland, Estonia and Lithuania. Similarly, men in the western countries have a higher risk of testicular cancer. According to the testicular dysgenesis syndrome (TDS) concept that suggests a link between risk of impaired semen quality and increased risk of testicular cancer, Spanish men would be expected to have a semen quality at a normal level because of their very low testis cancer risk. We therefore investigated 273 men from the Almeria region in the Southern Spain to test this hypothesis. The men delivered semen samples, underwent physical examinations, had a blood sample drawn and provided information on lifestyle and reproductive health parameters. The investigations took place from November 2001 to December 2002. Adjusting for effects of confounders, the median sperm concentration and total sperm count were 62 (95% confidence interval 47-82) million/mL and 206 (153-278) million, respectively. The median numbers of motile and morphologically normal spermatozoa assessed according to strict criteria were 59% (57-62%) and 9.4% (8.6-10.0%), respectively. The median total testosterone and calculated free androgen index were 28 nm (26-30) and 95 (88-103), respectively. Assuming that the investigated men, to a large extent, are representative of the population of young men the Southern Spain, the results show that these have normal semen quality and reproductive hormone levels as expected in a population with a low incidence of testicular cancer.

Recent adverse trends in semen quality and testis cancer incidence among Finnish men.

Impaired semen quality and testicular cancer may be linked through a testicular dysgenesis syndrome of foetal origin. The incidence of testis cancer has been shown to increase among Finnish men, whereas there is no recent publication describing temporal trends in semen quality. Therefore, we carried out a prospective semen quality study and a registry study of testis cancer incidence among Finnish men to explore recent trends. A total of 858 men were investigated in the semen quality study during 1998-2006. Median sperm concentrations were 67 (95% CI 57-80) million/mL, 60 (51-71) and 48 (39-60) for birth cohorts 1979-81, 1982-83 and 1987; total sperm counts 227 (189-272) million, 202 (170-240) and 165 (132-207); total number of morphologically normal spermatozoa 18 (14-23) million, 15 (12-19) and 11 (8-15). Men aged 10-59 years at the time of diagnosis with testicular cancer during 1954-2008 were included in the registry study, which confirmed the increasing incidence of testicular cancer in recent cohorts. These simultaneous and rapidly occurring adverse trends suggest that the underlying causes are environmental and, as such, preventable. Our findings necessitate not only further surveillance of male reproductive health but also research to detect and remove the underlying factors.

Overexpression of VEGF in testis and epididymis causes infertility in transgenic mice: evidence for nonendothelial targets for VEGF.

Vascular endothelial growth factor (VEGF) is a key regulator of endothelial growth and permeability. However, VEGF may also target nonendothelial cells, as VEGF receptors and responsiveness have been detected for example in monocytes, and high concentrations of VEGF have been reported in human semen. In this work we present evidence that overexpression of VEGF in the testis and epididymis of transgenic mice under the mouse mammary tumor virus (MMTV) LTR promoter causes infertility. The testes of the transgenic mice exhibited spermatogenic arrest and increased capillary density. The ductus epididymidis was dilated, containing areas of epithelial hyperplasia. The number of subepithelial capillaries in the epididymis was also increased and these vessels were highly permeable as judged by the detection of extravasated fibrinogen products. Intriguingly, the expression of VEGF receptor-1 (VEGFR-1) was detected in certain spermatogenic cells in addition to vascular endothelium, and both VEGFR-1 and VEGFR-2 were also found in the Leydig cells of the testis. The infertility of the MMTV-VEGF male mice could thus result from VEGF acting on both endothelial and nonendothelial cells of the male genital tract. Taken together, these findings suggest that the VEGF transgene has nonendothelial target cells in the testis and that VEGF may regulate male fertility.

MIP-1 alpha is a regulator of mitotic and meiotic DNA synthesis during spermatogenesis.

To find out whether macrophage inflammatory protein-1 alpha (MIP-1 alpha) has a role in the regulation of germ cell development, we studied its effects on spermatogenic stage-specific DNA synthesis in vitro. MIP-1 alpha increased the DNA synthesis of primitive type A2-4 spermatogonia and of premeiotic cells, whereas the DNA synthesis of more differentiated intermediate and type B spermatogonia was inhibited when cultured in the presence of MIP-1 alpha. An antibody against MIP-1 alpha cross-reacted with a protein of 15 kDa from every spermatogenic stage of rat seminiferous epithelium. Immunohistochemical studies with the same antibody revealed a complex pattern of MIP-1 alpha localization both in primitive and advanced spermatogenic cells. These observations suggest that MIP-1 alpha is a local regulator of mitotic and meiotic DNA synthesis.

Semen quality among Danish and Finnish men attempting to conceive. The Danish First Pregnancy Planner Study Team.

OBJECTIVE: To assess differences in semen quality between similar populations from Denmark and Finland. DESIGN: Comparison of semen quality between 221 Finnish men (of whom 115 had no proven fertility) and 411 Danish men with no proven fertility in two follow-up studies among normal couples trying to conceive. METHODS: In Finland male partners of couples without experienced infertility attempting to conceive were recruited through advertisements in local newspapers from 1984 to 1986. From 1992 to 1995 Danish men who lived with a partner and who had not attempted to achieve a pregnancy previously were recruited through their union when they discontinued birth control. All semen analyses were performed in accordance with the World Health Organization guidelines. RESULTS: Median sperm concentration, total sperm count and the percentage of morphologically normal spermatozoa were significantly higher among the Finnish men without proven fertility (104.0 million/ml, 304.0 million and 58% respectively) compared with the Danish men (53.0 million/ml, 140.8 million, and 41% respectively). Sperm concentration was 105.7% (95% confidence interval (CI) 58.1%-167.6%) and total sperm count was 127.4% (95% CI 71.4%-201.6%) higher among Finnish men without proven fertility than among Danish men after control for confounders. CONCLUSIONS: Some, but hardly all, of the observed difference in semen quality may be explained by differences in recruitment procedures, selection of the men and by methodological differences in semen analysis between the two countries. Also a birth cohort effect may explain some of the differences between countries as the Finnish men were recruited 11 years before the Danish men. Therefore, follow-up studies with identical recruitment and selection of men from the two countries are needed.

Electron microscopic immunolocalization of the 18 and 29 kilodalton secretory proteins in the mouse epididymis: evidence for differential uptake by clear cells.

In previous studies we reported the synthesis, secretion, and immunolocalization at the light microscopic level of two mouse epididymal proteins, MEP 7 and MEP 10 [Rankin et al. (1992b), Biol. Reprod., 46:747-766]. MEP 7 is the mouse homologue of the rat metalloproteins, AEG/D and E, and MEP 10 is the mouse homologue of the rat retinoic acid binding proteins, B and C. We now describe the immunolocalization of MEP 7 and MEP 10 in the mouse epididymis at the electron microscopic level. MEP 7 was localized in the Golgi apparatus, in small electron-lucent secretory vesicles, and on microvilli of the principal cells from the distal caput epididymidis to the cauda. The luminal contents were also immunoreactive in these regions of the epididymis. Although some gold particles were associated with the sperm surface, there was no selective concentration of these particles. In addition, MEP 7 was localized in large (600 nm) supranuclear endocytic vesicles and in infranuclear lysosomes. MEP 10 immunoreactivity was also seen on the microvilli of the principal cells of the distal caput and corpus and the luminal contents from the distal caput to the cauda epididymidis. There was no association of gold particles with the sperm surface. In contrast to MEP 7, there was no detectable MEP 10 immunoreactivity on the organelles of the principal cells involved in protein secretion or endocytosis. Clear cells also demonstrated immunoreactivity to MEP 7 and MEP 10. However, the intensity of immunolabeling, and the number of clear cells labeled, was greater with MEP 10 than MEP 7. In the case of MEP 7, the gold particles were located on the large supranuclear endocytic vesicles and on some infranuclear lysosomes, from the proximal corpus to the middle cauda, while in the case of MEP 10, gold particles were predominantly present in infranuclear lysosomes from the distal caput to the middle cauda. These results suggest that the principal cells are involved in both the secretion and endocytosis of MEP 7. The MEP 10 and MEP 7 proteins present in the lumen of the mouse epididymis are endocytosed from the lumen and degraded in the clear cells. However, the process of endocytosis by the clear cells of these two proteins appears to be different.

Morphometry of normal and teratozoospermic canine sperm heads using an image analyzer: work in progress.

Combining the traditional morphologic evaluation of spermatozoa with computer assisted image analysis adds randomness, objectivity, repeatability and accuracy to morphometric measurements. We collected semen from 10 fertile, normospermic dogs aged 1 to 7 yr and from 3 teratozoospermic breed-matched dogs. Sperm head morphology was examined in Giemsa-stained smears by light microscopy, using a computer-assisted image analyzer and by transmission electron microscopy. We found significant variation in sperm head area, length, width and degree of roundness among normospermic individual dogs, indicating that it would be necessary to examine many more dogs before the size and shape of normal dog spermatozoa could be determined. The normospermic dogs were used as controls for the teratozoospermic cases. Case 1: A 2-yr-old subfertile Cavalier King Charles Spaniel had semen with small and narrow-based sperm heads and a proximal cytoplasmic droplet in most of the spermatozoa. With the image analysis system, sperm heads were shown to be smaller and more oval than in normospermic dogs. The variatons in size and shape were similar in magnitude to those of control dogs. An examined infertile half-brother had similar semen quality. Case 2: A 3-yr-old Petit Basset Griffon Vendeen with 2 unsuccesfull matings exhibited spermatozoa with severe abnormalities. Measured by image analyzer, sperm heads were irregular in shape and very small in area. One of the two littermates examined had semen of the same quality as the case dog. Case 3: A 3-yr-old fertile Golden Retriever had semen with giant sperm heads in about 50% of spermatozoa. Image analyzing results revealed 2 populations of different sized sperm heads. Giant heads consisted of 52.2% of all spermatozoa. The results of the study reported here suggest that the image analysis technique may be useful in evaluating structural changes in sperm morphology, supplementing visual assessment that is used in conventional methods.

Three types of acrosomal aberrations of bull spermatozoa and their relation to fertility.

The effect of acrosomal aberrations of the spermatozoa of Finnish Ayrshire bulls on the corrected non-return rate within 60 days of the first 500 inseminations was studied. The material consisted of sperm samples examined by the artificial insemination societies. All samples had been accepted for use in artificial insemination. One Giemsa-stained slide was studied for each of the 95 bulls concerned. Samples showing distinct acrosomal defects were studied by electron microscopy. Three different types of acrosomal aberration were found. One was obviously associated with subfertility in all 6 bulls in which it was detected.

A cohort effect on serum testosterone levels in Finnish men.

OBJECTIVE: To investigate whether a population-level decline in serum testosterone exists in Finnish men. In comparison with other European populations, Finnish men have compared well in the studies of reproductive health (i.e. semen quality, incidence of cryptorchidism and testicular cancer); thus, we expected no significant cohort-dependent decrease in serum testosterone. METHODS: We analysed serum levels of testosterone, gonadotrophin and sex hormone binding globulin (SHBG) in 3271 men representing different ages (25-74 years) and birth cohorts within three large Finnish population surveys conducted in 1972, 1977 and 2002. RESULTS: Serum testosterone levels decreased (from 25.3 nmol/l in 25- to 29-year-old men gradually to 16.9 nmol/l in 70- to 74-year-old men), whereas SHBG and gonadotrophin levels increased with increasing age. In addition, a significant secular trend in testosterone (total and free), SHBG and gonadotrophin levels was observed with lower levels in more recently born age-matched men. Serum testosterone level decreased in men aged 60-69 years from 21.9 nmol/l (men born 1913-1922) to 13.8 nmol/l (men born 1942-1951). These decreases remained significant following adjustment for BMI. An age-independent birth cohort effect existed on reproductive hormones measured in the Finnish men. In concert with the lower free testosterone levels, we observed lower gonadotrophin levels, suggesting that while there may be detrimental changes at the gonad level, the hypothalamus-pituitary-axis is not responding appropriately to this change. CONCLUSIONS: The more recently born Finnish men have lower testosterone levels than their earlier born peers. This study offers no explanation for this substantial recent adverse development.

Epididymal maturation of the surface protein structure of mammalian spermatozoa.

The surface proteins of intact rat, ram, boar and bull spermatozoa from caput and cauda epididymidis were radioiodinated and analysed by SDS-polyacrylamide gel electrophoresis. The maturational changes in sperm surface protein patterns of the different species were compared to elucidate any possible common features or differences in the surface protein reorganization mechanism of mammalian spermatozoa during epididymal passage. New labelled proteins appeared on the surfaces of the rat and ram spermatozoa, whereas bull spermatozoa lost some surface protein components during maturation. The surface proteins of boar spermatozoa could be only weakly labelled by radioiodination and no distinct maturational change in the radioactivity patterns of labelled boar spermatozoa could be shown. The analysis of radioiodinated ram spermatozoa also revealed an obvious transformation of the labelled membrane lipids during epididymal transit. The conclusion is that the surface proteins of mammalian spermatozoa undergo some changes during epididymal maturation but there is no uniform mechanism in these changes common to all mammalian species.

Effect of seminal plasma and calcium on the stability of the surface protein composition of ejaculated bull spermatozoa.

Ejaculated bull spermatozoa were incubated in different media to elucidate the effect of seminal plasma and calcium on the stability of sperm surface compositions. The spermatozoa were radioiodinated prior to or after the incubation and the labelled proteins from the incubation media and spermatozoa were analysed by SDS-polyacrylamide gel electrophoresis. Seminal plasma detached efficiently sperm surface components. Even with concentration of 2% considerable amounts of the 44K and 17K proteins were released into the medium. The 17K proteins of seminal plasma were also adsorbed by sperm surfaces during incubation. The release of the 27K protein seemed to be dependent on calcium.

Radioiodination of surface proteins of bull spermatozoa and their characterization by sodium dodecyl sulphate--polyacrylamide gel electrophoresis.

Surface proteins of ejaculated bull spermatozoa were radioiodinated, solubilized and characterized by sodium dodecyl sulphate--polyacrylamide gel electrophoresis. The electron microscopic autoradiographs showed that the labelling was equally distributed to all parts of the spermatozoon and restricted to the sperm surface. The electrophoresis of solubilized radioactivity revealed 6 radioactive fractions with approximate molecular weights of 67000-69000, 47000-50000, 34000-37000, 25000-28000, and 14000-16000. The 6th fraction probably represented labelled lipids. The electrophoresis of radioiodinated seminal plasma proteins revealed only 2 radioactive protein peaks which coincided with the sperm surface protein fractions IV and V.

Changes in surface protein structure of bull spermatozoa during epididymal maturation.

The surface proteins of bull spermatozoa from caput and cauda epididymis were labelled by lactoperoxidase-catalyzed radioiodination and solubilized and analyzed by SDS-PAG-electrophoresis. The surface protein patterns of caput and cauda epididymal spermatozoa resembled each other but some distinct differences could be found. Caput epididymal spermatozoa revealed a protein peak with molecular weight of 15 000 - 18 000 daltons but this peak was not found on cauda epididymal spermatozoa. On caput epididymal spermatozoa the most intensely labelled protein peak was located between 90 000 and 100 000 daltons but on cauda epididymal spermatozoa the corresponding peak was only weakly labelled and had a molecular weight of 80 000 - 90 000 daltons. Surface protein with molecular weight of 42 000 - 47 000 daltons was dominating on cauda epididymal spermatozoa. The surface protein structure of cytoplasmic droplets did not drastically differ from that of epididymal spermatozoa.

Regional differences in distribution of surface proteins over the bull spermatozoa.

Surface-radioiodinated bull spermatozoa were ultrasonicated and fractionated by Percoll-gradient centrifugation. The different fractions obtained were solubilized and analyzed by SDS-polyacrylamide gel electrophoresis. Three fractions containing sperm heads, midpieces, and membranes and small fragments of the principal pieces were obtained. The electrophoresis revealed 5 main peaks representing the radioiodinated surface proteins with molecular weights of 80 000-90 000 (Ia), 68 000-75 000 (Ib), 42 000-47 000 (II), 33 000-37 000 (III) and 15 000-18 000 (V) from the intact spermatozoa as well as from each sperm fragment fraction. The major differences between fractions were in the relative magnitudes of the peaks. The peak II characteristically dominated in the head fraction, but was very small in the midpiece fraction. The results from the present study suggest that the peak II seen in the intact spermatozoa is mainly located on the head plasma membrane and that the differences in the sperm surface properties may be due to the uneven distribution or surface exposure of the proteins.

Tail stump sperm defect in Ayrshire bulls: morphogenesis of the defect.

Three cases of tail stump sperm defect have been presented in Finnish Ayrshire bulls. The semen samples of the bulls showed markedly reduced number of sperm with almost total absence of tails. Transmission and scanning electron microscopy of the testicular samples revealed the testicular origin of the defect. The anlagen of the implantation fossa, proximal and distal centrioles, connecting piece, nuclear ring, manchette and annulus were demonstrated, but the axoneme formation was completely blocked. A separate population of spermatids showed nuclear bending and was possibly destroyed by the phagocytic activity of Sertoli cells. The pedigree of the bulls supports the concept that the defect is inherited by a single recessive autosomal gene.

Ultrastructure of a tail stump sperm defect in an Ayrshire bull.

The sperm cells in the ejaculate of a sterile Ayrshire bull were studied by light microscopy as well as scanning and transmission electron microscopy. The spermatozoa showed an almost total lack of tails, whereas the heads appeared morphologically normal. The cytoplasmic residue at the caudal end of the head contained numerous membrane structures as well as mitochondria and incomplete elements of the proximal centriole and striated column. The basic disturbance appears to be a hereditary defect in the spermiogenesis.

Reassessment of sperm morphology of archival semen smears from the period 1980--1994.

Several reports have suggested that sperm counts of normal men have declined in many geographical regions during the last decades. Deterioration of sperm morphology has also been reported in some studies covering long sample collecting periods. The original semen analysis data of our semen laboratory from the period 1980--1994 showed a significant decline in the proportion of spermatozoa with normal morphology. The finding was, however, questioned because of changes in sperm morphology assessment criteria during the study period. In the present study 1745 smears were re-analysed to cover evenly the whole study period. The samples were examined in random order by using strict assessment criteria. Multiple linear regression analysis of the re-analysed data showed no effect of the year of sample delivery on sperm morphology between the years 1980 and 1994. However, there was a significant decline in the proportion of normal spermatozoa with later year of men's birth.