Hiroshi Harayama

Characteristics of Preimplantational Development of Porcine Parthenogenetic Diploids Relative to the Existence of Amino Acids In Vitro

Abstract The present study was designed to investigate the effects of amino acids on the in vitro development of porcine parthenogenetic diploids that were produced by electrostimulation (El-St) and cytochalasin B treatment of in vitro-matured oocytes. The culture medium for development, based on Whitten medium, contained 0.5 mg/ml of hyaluronic acid (mWM), and a two-step culture system in which 290 mOsmol before the 4-cell stage (48 or 72 h after El-St) and, subsequently, 256 mOsmol up to the blastocyst stage (mWMs) were used. In experiment 1, the diploids were cultured for 168 h in mWMs supplemented with 0.01–5 mg/ml of polyvinyl alcohol (PVA). In experiment 2, the diploids were cultured in mWMs containing 0.5 mg/ml of PVA (PVA-mWMs) for 0, 48, or 72 h and then cultured for 168 h after El-St in PVA-mWMs supplemented with essential amino acids for Eagle basal medium without glutamine (E-AA) and nonessential amino acids for minimum essential medium (NE-AA). The results showed that diploids can develop up to the blastocyst stage in mWMs including 0.05–5.0 mg/ml of PVA (49%–53% vs. 63%, P > 0.05), but the replacement of BSA with PVA alone could not support the expansion of blastocysts (11%–20% vs. 39%, P < 0.05) or their proliferation. The addition of both E-AA and NE-AA (E+NE-AA) to PVA-mWMs from the 1-cell stage resulted in severe inhibition of the development of diploids to the blastocyst stage. However, the addition of E+NE-AA to PVA-mWMs later than 48 or 72 h after El-St well supported the development of diploids to the blastocyst stage and supported the expansion of blastocysts. In experiments 3–5, which types of amino acids in E-AA inhibited the development of diploids during the first 48 h after El-St were determined. In experiment 6, the stimulatory effects of E-AA and/or NE-AA after the 4-cell stage were examined. The results of those experiments clearly showed that the presence of nonpolar E-AA, especially for valine, leucine, isoleucine, and methionine, during the first 48 h after El-St caused severe delay of the first division and inhibition of development beyond the 4-cell stage. The presence of NE-AA after the 4-cell stage produced a favorable condition for the expansion of blastocysts (33%), whereas the presence of E-AA increased the cleavage rates of the diploids after compaction and the total number of cells in the blastocysts (53.7 ± 2.7) and inner cell mass (12 ± 0.5). These findings indicate that the presence of nonpolar E-AA in a protein-free medium during the first 48 h causes the 4-cell block in porcine parthenogenetic diploids.

Capacitation-like alterations in cooled boar spermatozoa: assessment by the chlortetracycline staining assay and immunodetection of tyrosine-phosphorylated sperm proteins

This study was undertaken in order to characterize alterations occurring in cooled boar spermatozoa by a chlortetracycline (CTC) staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Spermatozoa were collected from 10 mature boars, washed and then resuspended in a Tris-citric acid-glucose (TCG) solution. The sperm suspensions were slowly cooled to 4 degrees C over 5 h and held for 2 days. Aliquots of the sperm suspensions were recovered before and after the cooling treatment and then used for the CTC staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Before the cooling treatment, almost all of the spermatozoa stained with CTC were characterized by uniform fluorescence over the whole head (an F pattern: uncapacitated spermatozoa). After the cooling treatment, however, significant higher percentages of spermatozoa exhibited a B pattern with a dark band of diminished fluorescence in the post acrosomal region and a relatively bright fluorescence in the acrosomal region (the pattern of capacitated spermatozoa). Coincidently, a 32 kDa tyrosine-phosphorylated protein appeared in the spermatozoa. However, these alterations occurring in the cooled spermatozoa were attenuated by the supplementation to the sperm suspensions with seminal plasma (20% (v/v)). Additionally, the same alterations were observed in the spermatozoa incubated in a capacitation-supporting medium (a modified Krebs-Ringer bicarbonate; mKRB) for 5 h. These results suggest that cooling could induce capacitation-like alterations in boar spermatozoa that were associated with the tyrosine phosphorylation of the 32 kDa sperm protein.

Stage-specific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig

The objective of this study was to investigate the effects of osmolarity of culture media on the development of porcine parthenogenetic diploids. Oocyte-cumulus-granulosa cell complexes were collected from ovaries and then in vitro-cultured for 48 h. The mature oocytes were subjected to a single electro-stimulation (El-St; 100 micros, 1500 V/cm), treated with 5.0 microg/ml Cytochalasin B for 4h and then cultured under various conditions as described below. In Experiment 1, the diploids were cultured for 168 h after El-St in modified Whittens medium with 256 mOsmol (mWM256), mKRB with 309 mOsmol, and mWM with 309 mOsmol (mWM309), in which the osmolarity was adjusted by addition of NaCl or mannitol, or by reduction of distilled water. In Experiment 2, the diploids were cultured in the five media used in Experiment 1 for the first 48 h, and then in mWM256 until 168 h after El-St. In Experiment 3, the diploids were cultured for the first 48 h in mWM with osmolarity adjusted from 256 to 330 mOsmol by addition of NaCl for the first 48 h and then in mWM256 until 168 h after El-St. In Experiment 4, the diploids were cultured in mWM with 290 mOsmol (mWM290) for the first period of 24, 48, or 72 h, and then in mWM256 until 168 h after El-St. In Experiment 5, after diploids were cultured in mWM290 for the first 48 h, the obtained 4-cell diploids were transferred to mWM with osmolarity adjusted from 200 to 310 mOsmol by addition of NaCl, then cultured until 168 h after El-St. All media were supplemented with 0.5mg/ml hyaluronic acid and 4.0mg/ml bovine serum albumin. The results obtained in Experiments 1-5 indicate that the osmolarity of a medium, but not the Na(+)/K(+) ratio, exerts effects on the development of diploids to the blastocyst stage. The change of osmolarity of the culture media after the 4-cell stage increased the rate of expanded blastocyst formation in porcine diploids. The optimal osmolarities of culture medium for the first 48 h after El-St (before the 4-cell stage) were 290 and 280-320 mOsmol, and those for the later period (after the 4-cell stage) were 256 and 220-270 mOsmol, respectively.

Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP-dependent hyperactivation in the spermatozoa of Japanese Black bulls.

The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen‐stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell‐permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine‐phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen‐stored spermatozoa were classified as having a high‐grade acrosome condition before incubation, sperm tyrosine‐phosphorylated proteins, including the 33‐kDa tyrosine‐phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41‐ and 33‐kDa sperm tyrosine‐phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine‐phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen‐stored sperm were classified as having a low‐grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine‐phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine‐phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP‐dependent hyperactivation for the spermatozoa of individual Japanese Black bulls. Mol. Reprod. Dev. 77:910–921, 2010.

Changes of PKA and PDK1 in the principal piece of boar spermatozoa treated with a cell-permeable cAMP analog to induce flagellar hyperactivation.

A cAMP‐induced protein tyrosine phosphorylation and flagellar hyperactivation are controlled via complicated signaling cascades in mammalian spermatozoa. For instance, these events seem to be regulated positively by the PKA‐mediated signaling and negatively by the PI3K/PDK1‐mediated signaling. In this article, we have shown molecular changes of PKA and PDK1 in cAMP analog (cBiMPS)‐treated boar spermatozoa in order to disclose possible roles of these kinases in protein tyrosine phosphorylation and hyperactivation. Ejaculated spermatozoa were incubated with cBiMPS, and then they were used for biochemical analyses of sperm kinases by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The first 30‐min incubation with cBiMPS highly activated PKA of the principal piece to the accompaniment of autophosphorylation on Thr‐197 of catalytic subunits. However, protein tyrosine phosphorylation and hyperactivation were fully induced in the sperm samples after the 180‐min incubation. A potentially active form of PDK1 (54/55‐kDa phospho‐PDK1) was detected in the principal piece of the spermatozoa during the 90‐min incubation. Another potentially active form (59‐kDa phospho‐PDK1) gradually increased during the same incubation period. However, the PDK1 suddenly became inactive by the dephosphorylation after the 180‐min incubation, namely coincidently with full induction of protein tyrosine phosphorylation and hyperactivation. Additionally, existence of PI3K‐dependently suppressing mechanisms for protein tyrosine phosphorylation was confirmed in the principal piece by pharmacological experiments with LY294002 and biochemical analyses with anti‐PI3K p85 antibodies. These findings suggest that dephosphorylation of PDK1 may be a molecular switch for enhancement of protein tyrosine phosphorylation and flagellar hyperactivation in boar spermatozoa. Mol. Reprod. Dev. 75: 1396–1407, 2008.

Testicular development in Chinese Meishan boars

The developmental process of the testis and age-related changes in the morphology of rete testicular spermatozoa were investigated in Meishan boars at 1 to 364 days of age. Testicular weight and the diameter of seminiferous tubules increased rapidly until 150 to 180 days of age. Leptotene stage spermatocytes, round spermatids and spermatozoa were first found in the section of seminiferous tubules at 30 to 45, 60 and 75 days of age, respectively. However, after 105 to 120 days of age, most rete testicular spermatozoa were morphologically normal. These results indicate that Meishan boars reach puberty as early as 75 days of age, though the testes acquire the ability to produce morphologically normal spermatozoa at about 120 days.

Relationship between cyclic AMP-dependent protein tyrosine phosphorylation and extracellular calcium during hyperactivation of boar spermatozoa

In mammalian spermatozoa, the state of protein tyrosine phosphorylation is modulated by protein tyrosine kinases and protein tyrosine phosphatases that are controlled via cyclic AMP (cAMP)‐protein kinase A (PKA) signaling cascades. The aims of this study were to examine the involvement of cAMP‐induced protein tyrosine phosphorylation in response to extracellular calcium and to characterize effects of pharmacological modulation of the cAMP‐induced protein phosphorylation state and calmodulin activity during hyperactivation in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell‐permeable cAMP analog) and CaCl2 at 38.5°C to induce hyperactivation, and then used for Western blotting and indirect immunofluorescence of phosphorylated proteins and for the assessment of motility. Both cBiMPS and CaCl2 were necessary for hyperactivation. The increase in hyperactivated spermatozoa exhibited a dependence on the state of cBiMPS‐induced protein tyrosine phosphorylation in the connecting and principal pieces. The addition of calyculin A (an inhibitor for protein phosphatases 1/2A (PP1/PP2A), 50–100 nM) coincidently promoted hyperactivation and cAMP‐induced protein tyrosine phosphorylation in the presence of cBiMPS and CaCl2. Moreover, the addition of W‐7 (a calmodulin antagonist, 2–4 µM) enhanced the percentages of hyperactivated spermatozoa after incubation with cBiMPS and CaCl2, independently of protein tyrosine phosphorylation. These findings indicate that cAMP‐induced protein tyrosine phosphorylation in the connecting and principal pieces is involved in hyperactivation in response to extracellular calcium, and that calmodulin may suppress hyperactivation via the signaling cascades that are independent of cAMP‐induced protein tyrosine phosphorylation. Mol. Reprod. Dev. 79: 727–739, 2012.

When does the sex ratio of offspring of the paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decrease: In the spermatozoa stage or at fertilization?

Recent animal experiments confirmed that paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases the sex ratio of offspring at birth without altering litter size. However, the timing of this decrease remained unclear. Male mice were administered TCDD at 7-12 weeks of age and mated with non-treated females. The Y-bearing/X-bearing sperm ratio was examined by real-time PCR and FISH methods, and the sex ratio of the 2-cell embryos collected from non-treated females that had been mated with TCDD-exposed males were investigated by nested PCR. The Y-bearing/X-bearing sperm ratio was not significantly decreased in the TCDD group. However, the sex ratio of the 2-cell embryos of the TCDD group was significantly lower than that of the control group. These results may have resulted from a decrease in fertility of Y-bearing sperm. Thus, the results of this study suggested that the sex ratio of the offspring was decreased at fertilization and not during the spermatozoa stage.

Biochemical characterization of sialoprotein "anti-agglutinin" purified from boar epididymal and seminal plasma.

Sialoprotein “anti‐agglutinin,” previously shown to inhibit sperm head‐to‐head agglutination, is found in both boar epididymal and seminal plasma. The present report characterizes anti‐agglutinin by mass spectrometry, by N‐terminal amino acid sequence analysis, and by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE) and Western blotting techniques to assess phosphate content of the molecule. Anti‐agglutinin had the SDS‐PAGE mobility of approximately 25 kDa. By electrospray ionization‐mass spectrometry, however, mass spectra of anti‐agglutinin were characterized by two major peaks (19,379–19,382 Da and 19,395–19,397 Da) and several minor peaks. Mass spectrometry of tryptic peptide fragments of deglycosylated anti‐agglutinin and amino acid sequence analysis revealed that the protein has a unique peptide‐mass fingerprinting of fragments (12,668 Da, 5,209 Da, 1,226 Da, and 1,168 Da) and a novel N‐terminal amino acid sequence (KTDDY AISGA KEEEF YDYME ELYAV), respectively. Additionally Western blot techniques, using commercially available monoclonal antibodies, were used to detect presence of phosphothreonine and phosphoserine substituents, but two different monoclonal antibodies did not detect phosphotyrosine. Moreover, treatment with two different alkaline phosphotases converted the molecule, as assessed by SDS‐PAGE and detection by silver stain, from the parent form of about 25 kDa to forms of approximately 19 kDa (similar to that assigned by mass spectrometry) and/or 15 kDa. Original antiserum generated toward, and reacting with native anti‐agglutinin, reacted only with 19 kDa form. These results are consistent with the conclusion that the native anti‐agglutinin may be a novel protein that is phosphorylated at serine and/or threonine residues. Mol. Reprod. Dev. 55:96–103, 2000.

Novel Approach for the Detection of the Vestiges of Testicular mRNA Splicing Errors in Mature Spermatozoa of Japanese Black Bulls

There is a serious problem with the reduction of male reproductive performance of the livestock in the world. We have a hypothesis that the splicing error-caused derivation of aberrant sperm motility-related proteins may be one of its causal factors. It is thought that fresh testicular tissues are necessary for the detection of splicing errors of the mRNA. However, it is difficult to obtain testicular tissues from a number of agriculturally important bulls by surgical methods, because such procedures may have deleterious effects on bulls’ reproductive performance. The aim of this study was to examine the usefulness of mRNA fragments collected from ejaculated spermatozoa as alternative analytical samples for detection of the splicing errors. In the first experiment, we characterized the alternative splicing and splicing error of bull testicular ADCY10 mRNA which coded the synthase of the regulatory molecule for sperm motility “cAMP”. In testes, the exon 11-lacking variant coding the truncated ADCY10 was derived by alternative splicing. However, splicing errors, which accompanied the frame shift in the second cyclase domain, were occasionally observed in the exon 11-lacking variant. This aberrant variant retained intronic nucleotides (4 bases, CCAG) connecting the initial part of exon 10 due to splicing errors and consequently yielded the cleavage site for a restriction enzyme (Cac8I) which recognized the nucleotide sequences (GCNNGC). In the second experiment, we recovered residual testicular mRNA fragments from ejaculated spermatozoa and observed the splicing error-caused derivation of the aberrant variant of ADCY 10. Ejaculated spermatozoa conserved mRNA fragments of the exon 11-lacking variant coding exons 9, 10, 12 and 13. Moreover, the above-mentioned aberrant variant of ADCY10 mRNA fragment was detectable by Cac8I digestion treatment using the sperm mRNAs. These results indicate the utility of sperm mRNA fragments for the detection of splicing errors in bull testicular mRNAs.