Chromosomal localization of the human protamine genes,PRM1 and PRM2, to 16p13.3 by in situ hybridization

Summary Protamines are sperm-specific proteins that replace histones in the nuclear chromatin of mature spermatozoa. A chromosomal localization of the genes coding for human protamines has been achieved by in situ hybridization. Two cDNA probes of 423 bp and 397 bp containing the entire coding sequence for human protamine 1 (HP1) and human protamine 2 (HP2), respectively, have been used. The genes, called PRM1 and PRM2, have been found, tightly linked, on band 16p13.3. Arguments are given for the existence of these two genes as single copies, PRM1 coding for the unique HP1 protamine and PRM2 coding for a precursor of several proteins belonging to the HP2 family.     

The genes for protamine 1 and 2 (PRM1 and PRM2) and transition protein 2 (TNP2) are closely linked in the mammalian genome.

The genes for two protamines (PRM1 and PRM2) and for two transition proteins (TNP1 and TNP2) have been characterized in several mammalian species. In the human, boar, and bull, the genes for PRM1, PRM2, and TNP2 are closely linked over a stretch of DNA 13-15 kb long. Although similar data are not yet available for the mouse and rat, our results suggest that the three genes are similarly linked in these species. The gene for TNP1 in all species studied is located on another chromosome.     

Association of CRISP2, CCT8, PEBP1 mRNA abundance in sperm and sire conception rate in Holstein bulls

The objective was to determine the association of mRNA expression of cystine rich secretary protein 2 (CRISP2), chaperonin containing T-complex protein 1, subunit 8 (CCT8), and phosphatidylethanolamine-binding protein 1 (PEBP1), in sperm of Holstein bulls with Sire Conception Rate (SCR) scores between −4 and +4. These proteins were involved in sperm capacitation and sperm-egg fusion. Samples of sperm obtained on a single day from Holstein bulls (N = 34) in a commercial AI centre were used to evaluate relative mRNA expression of CRISP2, CCT8, and PEBP1. The mRNA abundance of CRISP2 was positively correlated (r = 0.88; P < 0.002), CCT8 was negatively correlated (r = −0.87; P < 0.002), and PEBP1 was positively correlated (r = 0.83; P < 0.006) with SCR-scores. The means of CRISP2 mRNA abundance was greater among positive SCR-score bulls (2.5 to 8 fold), the means of CCT8 mRNA abundance was greater among the negative SCR-score bulls (9.5 to 3.5 fold), and the means of PEBP1 mRNA abundance was greater for the positive SCR-score bulls (5.4 to 7.7 fold). In multivariate regression models predicting SCR-scores, mRNA abundance of CCT8 was significantly associated with SCR-score in all models. In the presence of CRISP2 mRNA abundance in the model, the SCR score's predictability of PEBP1 was insignificant. However, in the absence of CRISP2 mRNA abundance in the model, the SCR-score's predictability of PEBP1 was significant. In multivariate regression models, CRISP2 and CCT8 mRNA expression in sperm accounted for 95% of the variance in Holstein bull's SCR-scores. In conclusion, Holstein bulls with greater CRISP2 and lower CCT8 mRNA expression in sperm had higher probabilities of siring calves.     

Association between mRNA abundance of functional sperm function proteins and fertility of Holstein bulls

Although the existence of a complex population of mRNA in sperm is well documented, its role has not been completely elucidated. The objective of this study was to determine the relationship of mRNA abundance of sperm specific proteins and sire conception rates (SCR; a fertility index) in Holstein bulls. Samples of sperm from a single collection from commercial Holstein bulls (N = 34) were used to evaluate relative mRNA expression of adenylate kinase (AK) 1, integrin beta (IB) 5, Doppel, nerve growth factor, tissue inhibitors of metalloproteinases (TIMP) 2, lactate dehydrogenase C 1, small nuclear ribonucleoprotein polypeptide N, outer dense fiber 2, and phospholipase C zeta (PLCz) 1 in sperm. With the exception of lactate dehydrogenase C 1 and outer dense fiber 2, the mRNA abundances of these proteins were greater (P < 0.05) for high fertility (> +2 to ≤ 4 SCR) bulls compared with average (≥ 2 to ≤ +2) and low fertility (> −2 to ≤ −4) bulls. Of all the multivariate regression models tested, a combination of AK1, IB5, TIMP2, small nuclear ribonucleoprotein polypeptide N, and PLCz1 accounted for 97.4% of the variance in SCR scores. In the absence of PLCz1, the combination of AK1, IB5, Doppel, nerve growth factor, TIMP, and small nuclear ribonucleoprotein polypeptide N accounted for 96.6% of the variance in SCR scores. In addition, immunocytochemistry confirmed that the sperm-specific protein markers evaluated in this study were present in sperm. In conclusion, frozen-thawed semen from bulls with higher AK1, IB5, TIMP, small nuclear ribonucleoprotein polypeptide N 2 and PLCz1 mRNA abundances in the sperm had greater correlations with sire fertility index and may possess greater probabilities of siring calves.     

Intestinal PTGS2 mRNA Levels,PTGS2 Gene Polymorphisms,and Colorectal Carcinogenesis

Background & Aims

Inflammation is a major risk factor for development of colorectal cancer (CRC). Prostaglandin synthase cyclooxygenase-2 (COX-2) encoded by the PTGS2 gene is the rate limiting enzyme in prostaglandin synthesis and therefore plays a distinct role as regulator of inflammation.

Methods

PTGS2 mRNA levels were determined in intestinal tissues from 85 intestinal adenoma cases, 115 CRC cases, and 17 healthy controls. The functional PTGS2 polymorphisms A-1195G (rs689466), G-765C (rs20417), T8473C (rs5275) were assessed in 200 CRC cases, 991 adenoma cases and 399 controls from the Norwegian KAM cohort.

Results

PTGS2 mRNA levels were higher in mild/moderate adenoma tissue compared to morphologically normal tissue from the same individual (P<0.0001) and (P<0.035) and compared to mucosa from healthy individuals (P<0.0039) and (P<0.0027), respectively. In CRC patients, PTGS2 mRNA levels were 8–9 times higher both in morphologically normal tissue and in cancer tissue, compared to healthy individuals (P<0.0001). PTGS2 A-1195G variant allele carriers were at reduced risk of CRC (odds ratio (OR) = 0.52, 95% confidence interval (95% CI): 0.28–0.99, P = 0.047). Homozygous carriers of the haplotype encompassing the A-1195G and G-765C wild type alleles and the T8473C variant allele (PTGS2 AGC) were at increased risk of CRC as compared to homozygous carriers of the PTGS2 AGT (A-1195G, G-765C, T8473C) haplotype (OR = 5.37, 95% CI: 1.40–20.5, P = 0.014). No association between the investigated polymorphisms and PTGS2 mRNA levels could be detected.

Conclusion

High intestinal PTGS2 mRNA level is an early event in colorectal cancer development as it occurs already in mild/moderate dysplasia. PTGS2 polymorphisms that have been associated with altered PTGS2 mRNA levels/COX-2 activity in some studies, although not the present study, were associated with colorectal cancer risk. Thus, both PTGS2 polymorphisms and PTGS2 mRNA levels may provide information regarding CRC risk.     

Sperm competition, sperm numbers and sperm quality in muroid rodents

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An "overall sperm quality" parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert.     

Timing of sperm transport, sperm penetration and cleavage in the rat

Times of sperm entry into the oviduct from the uterus, into the ampulla from the isthmus; of sperm penetration into oocytes, and of cleavage, were determined using three mating regions. Time intervals and their errors of estimation were calculated. Spermatozoa were first found in the isthmus of the oviduct no earlier than 15 minutes after coitus, but required four hours to ascend the oviduct to the ampulla. The rate of sperm arrival was equal to the rate of sperm penetration, i.e., about 3 sperms/hour. Time of cleavage in vivo was 20.6 hours after sperm penetration in ad libitum mated animals. In culture, oocytes cleaved at exactly the same time as in vivo. Delaying sperm arrival to the site of fertilization (by delaying mating) shortened the time interval between median time of sperm penetration and median time of cleavage. It was concluded that the time of cleavage of the oocyte reflects primarily the time of sperm penetration, but is also influenced by the postovulatory age of the oocyte.     

Diethylstilbestrol-treated adult rats with altered epididymal sperm numbers and sperm motility parameters, but without alterations in sperm production and sperm morphology

In this study, we characterized estrogenic effects of diethylstilbestrol (DES) on reproductive parameters in male rats to identify a minimal dose level that alters epididymal and sperm functions but has little or no effect on sperm production and/or spermatogenesis. Adult rats (five animals/group) received s.c. injections of 0.2 ml of corn oil containing DES at a rate of 1.0 mg, 200 microg, 40 microg, 8 microg, 1.6 microg, or 320 ng x rat(-1) x day(-1) for 12 days. The control group received corn oil only. DES effects were similar in the 8-microg group and higher dose groups and included significant (P < or = 0.05) reductions in 1) absolute and relative weights of the head and body of the epididymis (EP), tail of the EP, and seminal vesicle, 2) numbers of sperm in both regions of the EP, and 3) motility characteristics in sperm collected from the tail of the EP. Conversely, no significant changes were observed in relative testis weight, daily sperm production, spermatogenesis, seminiferous epithelial height in stage VII, and sperm morphology. All of the above parameters in the 1.6-microg group (except seminal vesicle weight) and 320-ng group were comparable to those of controls. Plasma testosterone (T) level was reduced to an almost undetectable level in the > or = 8-microg groups and to a very low level in the 1.6-microg group (0.35 vs. 2.36 ng/ml in controls or 320-ng group), but LH level was unaltered. In a parallel fertility study, males received DES at a rate of 40, 8, or 1.6 microg x rat(-1) x day(-1) for 12 days prior to and 12 days during cohabitation (1:1) with untreated females. Of the 15 females cohabited with treated males (5 females/dose), none in the 40-microg and 8-microg groups and 1 in the 1.6-microg group formed a copulatory plug and delivered 8 pups, in contrast to 5/5 copulatory plugs and 13-15 pups/litter in the controls. DES at a rate of 8 microg x rat(-1) x day(-1) for 12 days reduced EP weights, sperm numbers in the EP, and sperm motility patterns but caused minimal to no alterations in daily sperm production, spermatogenesis, or sperm morphology. Factors other than T, or in addition to lower T, may be responsible for DES-induced reproductive disorders (despite lower T, sperm contents and sperm motility patterns in the EP were normal in the 1.6-microg group). Deficits in EP sperm functions and/or sexual behavior (as evident from absence of copulatory plugs) probably accounted for reduced fertility in treated males.     

SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD,RBD, and S2

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Mapping of PRM1 to human chromosome 16 and tight linkage of Prm-1 and Prm-2 on mouse chromosome 16

The protamines are small, arginine-rich nuclear proteins that replace histones and transition proteins late in the haploid phase of spermatogenesis in mammals. The two mouse genes encoding protamines--Prm-1 and Prm-2--have been molecularly cloned and mapped to mouse chromosome 16 (MMU 16). A cDNA clone of mouse Prm-1 that hybridized to the corresponding human gene was used to analyze a panel of somatic cell hybrids made between human lymphoblasts and the E36 hamster cell line. The human gene, which we have designated PRM 1, was syntenic with human chromosome 16 (HSA 16) and discordant with all other human chromosomes. Linkage analysis in the mouse was accomplished using the backcross (Czech II x BALB/c Pt) x Czech II to map Prm-1 and Prm-2 to a position near the 5' terminus of MMU 16. No recombination between Prm-1 and Prm-2 was observed among 89 progeny of the Czech II x BALB/c cross or among 94 progeny of the backcross (CBA/J x BALB/cJ) x BALB/cJ, demonstrating that the two loci are separated by less than 1.6 cM on MMU 16. This tight linkage may be of functional significance, as Prm-1 and Prm-2 are among a limited number of genes known to be expressed postmeiotically in male haploid germ cells.