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E. E. Montamat, J. Moreno and A. Blanco

Summary. Branched-chain amino acid aminotransferase (l-leucine:2-oxoglutarate aminotransferase, EC 2.6.1.6) activity was determined in several tissues of the mouse. Testis homogenates presented a specific activity very close to that of heart extracts which were the most active. Enzyme activity was detectable in testes from 5-day-old mice and increased steadily during development to reach a maximum at the 20th day of life. The transaminase was present in the cytosol of testicular homogenates and also associated, probably in the matrix, with a special type of mitochondria present in spermatozoa and gametogenic cells. The enzyme from testis is active against the three branched-chain amino acids and catalyses the reaction in both directions. Highest activity and lowest K m were obtained with l-leucine. Activity with l-valine was the lowest. The enzyme from the mitochondrial fraction showed identical properties to that from the soluble phase. The possible participation of this aminotransferase in a shuttle system transferring reducing equivalents from cytoplasm to mitochondria is postulated.

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L. J. BATTELLINO, J. SABULSKY and A. BLANCO

Summary.

Total lactate dehydrogenase-specific activity and concentration of isoenzymes were determined on extracts of rat uterus at different stages of pregnancy, during the oestrous cycle and after ovariectomy.

Total enzymatic activity increased during pregnancy. The rise was evident by the 5th day and continued steadily until the 14th day, when it attained maximal values. These high values were maintained up to the end of pregnancy and fell to the level of controls 4 days after parturition.

The changes in enzyme activity were entirely due to variations in the amount of Isoenzymes 5 (A4) and 4 (A3B1). The other three molecular forms were not modified

Total specific activity and Isoenzymes 5 and 4 were diminished 20 days after ovariectomy.

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J Blanco-Rodriguez, C Martinez-Garcia and A Porras

In the seminiferous epithelium, both DNA synthesis and apoptosis occur at equivalent stages in various species, with apoptosis taking place mainly at the same stages as DNA replication in the second, third and fourth spermatogonial generations. As preservation of the cellular associations found at these stages may have some functional significance, it is important to determine whether there is a correlation between these cellular events. In this study, pairs of immunoperoxidase-stained adjacent testis sections from rats, mice, rabbits and cats in which either bromodeoxyuridine incorporated into the newly synthesized DNA strand (BrdU labelling) or DNA 3' end labelling of the apoptotic DNA fragments (TUNEL assay) were detected were compared. In addition, both events were analysed in double-labelled sections. These two methods revealed a clear correlation between the occurrence of DNA replication in the second to fourth generations of spermatogonia and most physiological apoptosis taking place in both spermatogonia and spermatocytes in the three different mammalian orders (Rodentia, Lagomorpha and Carnivora). This correlation may result from the synchronization of mitotic spermatogonial and meiotic spermatocyte cell cycle checkpoints operating at these stages.

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C. Burgos, N. M. Gerez de Burgos, C. E. Coronel and A. Blanco

Summary. The activity of lactate dehydrogenase (EC 1.1.1.27) in normal human sperm lysates and in human heart and liver homogenates was determined by using a variety of 2-oxoacids as substrates. Sperm preparations were active with pyruvate, 2-oxobutanoate, 2-oxopentanoate and 2-oxohexanoate, while heart and liver extracts utilized only pyruvate and 2-oxobutanoate. Selective staining after gel electrophoresis indicated that the fraction corresponding to lactate dehydrogenase C4, the sperm-specific isoenzyme, was responsible for the utilization of substrates with a linear chain of 3 to 6 carbon atoms. The use of 5 mM 2-oxohexanoate allowed the selective determination of isoenzyme C4 in preparations containing different lactate dehydrogenase molecular forms.

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B Fernandez-Fuertes, A Blanco-Fernandez, C J Reid, K G Meade, S Fair and P Lonergan

This study tested the hypothesis that sperm sialic acid (Sia) is required to reach the site of fertilization, and that successful fertilization requires recognition of Sia from both the sperm and oocyte to occur. In addition, it has recently been reported that Siglecs (Sia-binding-immunoglobulin-like lectins) are present on the sperm surface. Thus, the possibility that the recognition of oocyte Sia was sperm-Siglec-mediated was also addressed. Sperm exposed to neuraminidase (NMase) exhibited lower overall and progressive motility, which translated to a decreased ability to swim through cervical mucus from cows in oestrus. In addition, when either sperm or cumulus–oocyte complexes (COCs) were treated with NMase, a decrease in cleavage and blastocyst rate was observed. However, incubation of sperm with increasing concentrations of anti-Siglec-2, -5, -6 and -10 antibodies prior to fertilization had no effect on their fertilizing ability. Interestingly, treatment with NMase increased the number of sperm bound to the ZP but also the rate of polyspermic fertilization. Flow cytometry analysis revealed no differences in the percentage of capacitated or acrosome-reacted sperm. These results suggest that Sia are required to reach the site of fertilization but need to be removed for sperm–oocyte interaction. However, fine regulation is needed to avoid abnormal fertilization which can lead to impaired embryo development.

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K Wagoner, G Sanchez, A-N Nguyen, G C Enders and G Blanco

Two catalytic isoforms of the Na,K-ATPase, α1 and α4, are present in testis. While α1 is ubiquitously expressed in tissues, α4 predominates in male germ cells. Each isoform has distinct enzymatic properties and appears to play specific roles. To gain insight into the relevance of the Na,K-ATPase α isoforms in male germ cell biology, we have studied the expression and activity of α1 and α4 during spermatogenesis and epididymal maturation. This was explored in rat testes at different ages, in isolated spermatogenic cells and in spermatozoa from the caput and caudal regions of the epididymis. Our results show that α1 and α4 undergo differential regulation during development. Whereas α1 exhibits only modest changes, α4 increases with gamete differentiation. The most drastic changes for α4 take place in spermatocytes at the mRNA level, and with the transition of round spermatids into spermatozoa for expression and activity of the protein. No further changes are detected during transit of spermatozoa through the epididymis. In addition, the cellular distribution of α4 is modified with development, being diffusely expressed at the plasma membrane and intracellular compartments of immature cells, finally to localize to the midregion of the spermatozoon flagellum. In contrast, the α1 isoform is evenly present along the plasma membrane of the developing and mature gametes. In conclusion, the Na,K-ATPase α1 and α4 isoforms are functional in diploid, meiotic and haploid male germ cells, α4 being significantly upregulated during spermatogenesis. These results support the importance of α4 in male gamete differentiation and function.

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S Gimeno-Martos, M González-Arto, A Casao, M Gallego, J A Cebrián-Pérez, T Muiño-Blanco and R Pérez-Pé

This study was based on the assumption that steroid hormones present in the female genital tract may have a rapid effect on ram spermatozoa by interaction with specific surface receptors. We demonstrate the presence of progesterone (PR) and estrogen (ER) receptors in ram spermatozoa, their localization changes during in vitro capacitation and the actions of progesterone (P4) and 17β-estradiol (E2) on ram sperm functionality. Immunolocalization assays revealed the presence of PR mainly at the equatorial region of ram spermatozoa. Western blot analyses showed three bands in ram sperm protein extracts of 40–45 kDa, compatible with those reported for PR in the human sperm membrane, and both classical estrogen receptors (66 kDa, ERα and 55 kDa, ERβ). ERα was located in the postacrosomal region of all the spermatozoa and ERβ on the apical region of 63.7% of the cells. The presence of ERβ was correlated with the percentage of non-capacitated spermatozoa evaluated by chlortetracycline staining (R = 0.848, P < 0.001). This significantly decreased after in vitro capacitation and nearly disappeared when acrosome reaction was induced. The addition of P4 and E2 before in vitro capacitation resulted in a higher (P < 0.001) acrosome-reacted sperm rate compared with the control (13.0%), noticeably greater after 3 h and when added to a high-cAMP medium (37.3% and 47.0% with E2 and P4, respectively). In conclusion, the results of this study demonstrate for the first time that ovine spermatozoa have progesterone and estrogen receptors and that both steroid hormones are related with the induction of the acrosome reaction.

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N. M. Gerez de Burgos, C. Burgos, C. E. Coronel, A. Bertarelli de Camusso, T. Pigini and A. Blanco

Summary. The activity of lactate dehydrogenase isoenzyme C4 was determined on 90 human semen samples. The correlation between the isoenzyme activity and sperm count and motility was good (r = 0·74 for values of U/ml semen against sperm count).