Effect of mitochondrial DNA (mtDNA) type on pregnancy rate after embryo transfer in rats.

Because the mtDNA is maternally inherited, embryos resulting from matings or artificial inseminations have the same type of mtDNA as that of the mother. However, when embryos are transferred the mtDNA of the embryos may be different to that of the recipient and this may interfere with maternal recognition and the establishment of pregnancy. This study was done to determine whether differences in the mtDNA between embryos and recipients would influence the survival to term of transferred embryos.A total of 1,220 rat embryos were recovered from non-superovulated donors of known mtDNA type. The number and distribution of developmental stages of embryos collected from 51 rats of mtDNA type A (n = 595) were not different (P>0.05) from those collected from 50 rats of mtDNA type B (n = 625). The overall pregnancy rate after transfer of embryos to pseudopregnant rats was 54% (26 48 ). The pregnancy rate was not affected (P>0.05) by the type of mtDNA of the recipient or of the embryo, and the interaction between mtDNA type of embryos and recipients was also not significant (P>0.05). Embryonic survival to birth was low (78 622 , 12.5%) but was not affected (P>0.05) by the type of mtDNA of the recipient (A = 28 250 ; B = 50 372 ) or of the embryo (A = 41 306 ; B = 37 316 ). Survival of pups to weaning was affected by the type of mtDNA of the embryo (P < 0.01) but not by the type of mtDNA of the recipient (P>0.05) and the interaction between mtDNA type of embryos and recipients was also not significant (P>0.05). More pups (P < 0.005) derived from donor rats of mtDNA type A (34 41 ) survived to weaning age than pups from donor rats of mtDNA type B (18 37 ). These results indicate that differences in the type of mtDNA between embryos and recipients do not interfere with establishment of pregnancy in pseudopregnant recipients.

Cytoplasmic inheritance and its effects on development and performance.

In contrast to nuclear inheritance, cytoplasmic inheritance in mammals is derived mostly, if not exclusively, from the maternal line. Mitochondria, and their DNA molecules (mtDNA), are the genetic units of this method of inheritance. Mammalian mtDNA codes for 13 enzymes used in the mitochondrial energy-generating pathway, oxidative phosphorylation, 22 tRNAs and two rRNAs. Although all transcripts of mtDNA and their translational products remain in the mitochondria, most proteins used in mitochondria are from nuclear DNA and are imported after synthesis on cytoplasmic ribosomes. Spermatozoa introduce a small number of mitochondria into the cytoplasm of the egg at fertilization, which appear to be digested soon after penetration. Although the paternal contribution of mtDNA to the offspring is not believed to occur in mammals, some interspecific crosses have suggested that it does occur. Experiments with animals derived from reconstituted embryos, using nuclear or cytoplasmic transplantations, suggest that nuclear-mitochondrial interactions are important but not essential in the survival and replication of exogenous mitochondria introduced into the egg. As the levels of heteroplasmy varied in several tissues of animals derived from reconstituted embryos, it is suggested that differential partitioning of mitochondria occurs during embryogenesis. Mitochondrial morphology changes substantially during oogenesis and throughout early cleavage stages. Somatic morphology and normal replication patterns are regained at the blastocyst stage. In pig oocytes and embryos, mitochondria aggregate and are closely associated with endoplasmic reticulum, lipid granules and large vesicles. Although the direct correlation of mitochondrial genes with reproductive traits is still unclear, some human degenerative diseases and performance traits in cattle can be related directly to specific mtDNA polymorphisms. In pigs, reciprocal-cross comparisons have indicated greater offspring parent similarity with dam than sire for lean:fat ratio. A difference was also observed for oxygen consumption and oxidative phosphorylation, but not for anaerobic energy metabolism, in a pig reciprocal-cross experiment. Information on the transmission of mtDNA and its effects on performance will have many implications not only for our understanding of mitochondrial genetics but also for the increased productivity of animals. There are also potential ramifications to the animal cloning industry.

Temporal gene expression is restored concomitantly with germ cells in the experimentally regressed rat testis.

The present study was designed to examine the effect of hypophysectomy and subsequent testosterone administration on germ cell numbers and germ cell- and Sertoli cell-specific mRNA levels in adult rats. Rats were hypophysectomized and 4 weeks later received 24-cm testosterone-containing polydimethylsiloxane (PDS) implants. Sham-hypophysectomized rats received an empty PDS implant. At 0 and 3 days, and at 1, 2, 4, and 8 weeks, rats were killed. One testis from each rat (n = 4/group) was used to prepare total RNA; the other testis was used to enumerate stage VII-VIII germ cells. cDNA probes for germ cell and Sertoli cell products were used to monitor germ cell- and Sertoli cell-specific mRNAs on Northern blots. Four weeks after hypophysectomy (0 days), preleptotene and pachytene spermatocytes and round and elongating spermatids were reduced in number to 54%, 12%, 1%, and 0%, respectively, of the control values. Testosterone administration caused a time-dependent increase in germ cell numbers; after 8 weeks of testosterone treatment, preleptotene and pachytene spermatocytes and round and elongating spermatids were 75%, 79%, 74%, and 22%, respectively, of control values. Lactate dehydrogenase-C, phosphoglycerate kinase-2, protamine-1, and sulfated glycoprotein-2 mRNA levels (on a per micrograms RNA basis) were 34%, 34%, less than 1%, and 580% of control values, respectively, 4 weeks after hypophysectomy and 79%, 87%, 61%, and 192% of control values, respectively, after 8 weeks of testosterone treatment. Pachytene spermatocyte and round spermatid numbers increased, while Sertoli cell sulfated glycoprotein-2 mRNA levels decreased, with respect to 4 week hypophysectomy values, as early as 3 days after implantation of testosterone capsules. In contrast, germ cell (lactate dehydrogenase-C, phosphoglycerate kinase-2, and protamine-1) mRNA levels increased to the greatest extent between 1-4 weeks after the start of testosterone treatment and, after a short lag period, reflected increases in germ cell type and number. The results indicate that cell-specific mRNAs appear concomitantly with germ cell reappearance in a time-dependent manner in the testes of testosterone-treated hypophysectomized adult rats.

DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells.

Genomic methylation patterns are established during maturation of primordial germ cells and during gametogenesis. While methylation is linked to DNA replication in somatic cells, active de novo methylation and demethylation occur in post-replicative spermatocytes during meiotic prophase (1). We have examined differentiating male germ cells for alternative forms of DNA (cytosine-5)-methyltransferase (DNA MTase) and have found a 6.2 kb DNA MTase mRNA that is present in appreciable quantities only in testis; in post-replicative pachytene spermatocytes it is the predominant form of DNA MTase mRNA. The 5.2 kb DNA MTase mRNA, characteristic of all somatic cells, was detected in isolated type A and B spermatogonia and haploid round spermatids. Immunobolt analysis detected a protein in spermatogenic cells with a relative mass of 180,000-200,000, which is close to the known size of the somatic form of mammalian DNA MTase. The demonstration of the differential developmental expression of DNA MTase in male germ cells argues for a role for testicular DNA methylation events, not only during replication in premeiotic cells, but also during meiotic prophase and postmeiotic development.

DNA polymerase-beta and poly(ADP)ribose polymerase mRNAs are differentially expressed during the development of male germinal cells.

We have examined the steady-state mRNA levels in spermatogenic cells of two nuclear enzymes that appear to be involved in DNA repair, DNA polymerase-beta (pol-beta) and poly(ADP)ribose polymerase (PADPRP). Two pol-beta mRNAs of 1.3 kb and 1.4 kb were detected in extracts from mouse testes. In leptotene/zygotene spermatocytes a low level of the 1.4-kb mRNA was observed. Both pol-beta mRNAs were found in meiotic pachytene spermatocytes, with the 1.3-kb form being more abundant. In contrast, the 1.4-kb form was more abundant in haploid round spermatids. Polysome gradient analyses indicated that the two pol-beta mRNAs were predominantly present in the nonpolysomal fractions of spermatocytes. In round spermatids, a larger fraction of the 1.4-kb pol-beta mRNA was associated with polysomes, correlating well with the higher levels of pol-beta enzyme detected during spermiogenesis. The pattern of PADPRP mRNA expression differed from the expression of pol-beta mRNA. The two PADPRP mRNAs of 3.7 and 3.8 kb were present in type A and type B spermatogonia, reached their highest levels in pachytene spermatocytes, and were greatly reduced in haploid round and elongating spermatids. Most of the pachytene spermatocyte PADPRP and mRNAs were present in polysomes, whereas a greater percentage of PADPRP mRNAs in round spermatids were detected in the nonpolysomal fractions. This finding correlates with the immunocytochemical nuclear localization of this enzyme in pachytene spermatocytes. These data demonstrate that different developmental patterns of mRNA expression and translational regulation exist for the pol-beta and PADPRP mRNAs during differentiation of male germinal cells.

Endocrine changes in beef heifers superovulated with follicle-stimulating hormone (FSH-P) or human menopausal gonadotropin.

The effects of superovulatory treatment (FSH-P vs human menopausal gonadotropin, HMG) and of route of administration (i.m. vs. i.v.) of prostaglandin F2 alpha (PGF2 alpha) on hormonal profiles were determined in 32 Angus x Hereford heifers. Heifers were superstimulated with either FSH-P (total of 26 mg) or HMG (total of 1,050 IU) beginning on d 9 to 12 of an estrous cycle and PGF2 alpha (40 mg) was administered at 60 and 72 h after the beginning of superovulatory treatments. Heifers were artificially inseminated three times at 12-h intervals beginning 48 h after PGF2 alpha treatment. Blood serum samples were collected immediately before treatments began, at 12-h intervals during the first 60 h, each 4 h during the next 96 h, and each 12 h until day of embryo collection. Concentrations of LH and FSH were not affected by hormone treatments, route of PGF2 alpha injection, or interactions between them. Estradiol-17 beta (E2-17 beta) levels were higher (P less than .05) in HMG- than in FSH-P-treated heifers 60 h after gonadotropin treatment. Peak concentration of E2-17 beta occurred earlier (P less than .05) in HMG- than in FSH-P-treated heifers and earlier in heifers injected with PGF2 alpha i.m. than in those injected i.v. Progesterone concentrations were not influenced by treatment or route of PGF2 alpha administration, but were affected (P less than .01) by the interactions between treatment and route of PGF2 alpha administration. Progesterone declined to basal levels earlier in the FSH-P- than in the HMG-treated heifers.(ABSTRACT TRUNCATED AT 250 WORDS)

junD mRNA expression differs from c-jun and junB mRNA expression during male germinal cell differentiation.

The members of the jun family of protooncogenes (junB, c-jun, and junD) share a high degree of sequence homology and function as transcriptional regulators. Here we compare the pattern of junD mRNA expression during spermatogenesis to that of junB and c-jun (Alcivar et al.: J Biol Chem 265:20160-20165, 1990). junD transcripts are present at high levels in total RNA obtained from both prepuberal and adult intact testes, with the highest levels at stages containing predominantly premeiotic and postmeiotic germ cells. Analyses of cells isolated from testes of 8-day-old mice indicate that the level of the 1.8 kb junD mRNA is higher in type B spermatogonia than in type A spermatogonia. In testes of 17-day-old mice, the highest junD mRNA levels are detected in preleptotene spermatocytes compared to leptotene/zygotene and prepuberal pachytene spermatocytes. In cells from adult testes, the junD mRNA levels are higher in postmeiotic round spermatids and residual bodies/cytoplasts than in meiotic pachytene spermatocytes. An additional junD transcript of about 1.6 kb is detected in postmeiotic cells. Analyses of polysomal and nonpolysomal RNAs prepared from isolated testicular cells indicate that in early meiotic cell types the junD transcript is more efficiently loaded onto polysomes than in later cell types. In summary, the pattern of expression of junD differs from that of junB and c-jun during spermatogenesis most notably in that 1) junD mRNA levels do not increase following dissociation of testicular cells and 2) in contrast to the nearly undetectable levels of junB and c-jun mRNAs in adult postmeiotic testicular cells, high levels of junD mRNAs are seen.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA methylation and expression of the genes coding for lactate dehydrogenases A and C during rodent spermatogenesis.

The testis chromatin undergoes profound structural alterations and functional changes during spermatogenesis. Changes in DNA methylation have been correlated with gene expression in a number of systems, but the relationship between methylation and gene expression for testicular genes is unclear. To address this question, DNA methylation patterns and mRNA expression for a somatic form of lactate dehydrogenase (LDH), LDH-A, were compared with those of the testis-specific form, LDH-C, in preparations from testes of prepubertal and sexually mature mice, from isolated testicular cells, and from somatic tissues. At specific sites, LDH-A was less methylated in adult testis than in spleen DNA; the decreased methylation in the testicular DNA occurred as early as type A spermatogonia. In contrast, DNA methylation patterns for LDH-C did not differ between spleen and testis DNAs. In Northern blots, the levels of LDH-A transcripts were low in total testis RNA obtained from 6-12-day-old mice, and in type A and B spermatogonia from 8-day-old mice. LDH-A mRNA levels increased gradually in testes from 16-45-day-old mice. LDH-C transcripts were first detectable in the testes of 12-day-old mice and increased as spermatogenesis proceeded. Both LDH-A and LDH-C mRNA levels were low in preleptotene spermatocytes and leptotene/zygotene spermatocytes and increased substantially in pachytene spermatocytes and round spermatids. Reduced levels of LDH-A and LDH-C mRNAs were found in the residual bodies/cytoplasts fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased levels of junB and c-jun mRNAs in male germ cells following testicular cell dissociation. Maximal stimulation in prepuberal animals.

We have examined the relative transcript levels of the junB and c-jun proto-oncogenes during development of the mouse testis. junB and c-jun mRNA levels are low in total RNA from intact immature or mature testes. Dissociation of testicular cells, however, increases the levels of junB and c-jun mRNAs, with higher increases in the dissociated cells from testes of 8-day-old mice than from 17-day-old or sexually mature mice. These differences in junB and c-jun mRNA levels localize to specific cell types. In testes from 8-day-old mice, the mRNA levels for both proto-oncogenes are higher in type B spermatogonia and in the interstitial cell fraction than in type A spermatogonia. In testes of 17-day-old mice, the highest mRNA levels for both proto-oncogenes are seen in preleptotene spermatocytes and interstitial cells, with decreasing levels in leptotene/zygotene spermatocytes and prepuberal pachytene spermatocytes. junB and c-jun mRNAs are nearly undetectable in pachytene spermatocytes, round spermatids, and residual bodies/cytoplasts. The increased junB mRNA levels originate not only from the expected 2.1-kilobase transcript but from a more slowly migrating transcript of about 2.3 kilobases. RNase H analysis demonstrates that this migration change was due to an increase in mRNA polyadenylation. The low levels of junB and c-jun mRNAs in intact testes and the much higher levels in isolated cells from identical testes suggest that the disruption of cell-to-cell contact increases the amount of junB and c-jun transcripts in specific cells of the testis. Coupled with this increase, structural changes are seen with the junB mRNA.

Stage- and cell-specific expression of the ornithine decarboxylase gene during rat and mouse spermatogenesis.

Ornithine decarboxylase (ODC) is an enzyme that has been shown to be induced in the growth, differentiation and proliferation of cells. We have used a cDNA probe to determine ODC mRNA levels in different stages of the cycle of rat and mouse seminiferous epithelium. For Northern and slot-blot hybridizations, RNA was isolated from microdissected staged seminiferous tubules. Cell-specific localization of ODC mRNA was studied by in situ hybridization. In the rat, in situ hybridization showed increasing mRNA levels during prophase of meiosis with the highest mRNA levels seen in late pachytene spermatocytes and step 3-5 spermatids. In the mouse, the mRNA levels increased in a similar fashion and the highest mRNA levels were found in step 1-8 spermatids. In the rat, Northern blot hybridizations revealed three molecular sizes of ODC mRNA: 2.2, 2.7 and 1.6 kb. The levels of all molecular sizes were highest in stages VII-VIII, and the lowest mRNA levels were seen in stage I of the seminiferous epithelial cycle. The level of the 2.2 kb transcript was low during stages XIII-I. In the mouse, the Northern blot hybridizations also showed three molecular sizes of ODC mRNA: 2.2 and 2.7 kb and very low levels of 1.6 kb transcript. The levels of the transcripts were steady throughout the cycle. In the mouse, the 2.2 kb transcript was more abundant than the 2.7 kb transcript indicating a species difference between rat and mouse in the usage of the two polyadenylation signals within the ODC gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse.

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.

DNA methylation and demethylation events during meiotic prophase in the mouse testis.

The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.

Developmental and differential expression of the ornithine decarboxylase gene in rodent testis.

Ornithine decarboxylase (ODCase) is the first and rate-limiting enzyme in the polyamine biosynthetic pathway and it is androgen regulated in the mouse. The expression of ODCase transcripts during testicular development was examined by Northern blot analysis with a mouse ODCase cDNA probe. Total RNA was isolated from the testes of prepubertal mice at 6, 8, 12, 16, 18, 20, 22, and 30 days of age, from enriched populations of germinal cells obtained from the testis of immature (8 days old) and mature (45 days old) mice and from several mouse somatic tissues. The level of the two ODCase transcripts (2.2 and 2.7 kilobases) was low but detectable in the testes of 6- to 16-day-old mice and increased substantially as the first spermatogenic wave proceeded into spermiogenesis. The low ODCase mRNA levels observed in prepubertal mouse testes were confirmed with RNA samples obtained from enriched germ cell populations of type A and type B spermatogonia and interstitial cells obtained from Day 8 mouse testes. In agreement with the developmental studies, ODCase mRNA levels increased substantially in enriched populations of pachytene spermatocytes, round spermatids, and residual bodies/cytoplasts isolated from mature testes. Similar results were obtained by in situ hybridization of sections of rat testes. Reduced levels of ODCase transcripts were detected in RNA obtained from cultured mouse Sertoli cells obtained from the testes of 21-day-old mice and in RNA from liver, brain, heart, spleen, seminal vesicle, and aorta. In contrast, ODCase transcript levels from kidneys of male mice were as high as those detected in testis RNA. Substantial levels of ODCase mRNAs were also found in the epididymis. Analysis of polysome gradients prepared from total testis extracts revealed a distribution of ODCase mRNA in both nonpolysomal and polysomal fractions of the gradient, suggesting that ODCase is translationally regulated in the mouse testis.

Mitochondrial gene expression in male germ cells of the mouse.

Inheritance of the mitochondrial genome is known to be exclusively maternal. To determine whether the loss of paternal mitochondria could be due to a deficiency of RNA in the spermatozoal mitochondria, the expression of mitochondrial genes was studied in testicular cells at various stages of spermatogenesis and in epididymal spermatozoa. The presence of mitochondrial transcripts was examined by Northern blot analysis using probes for the following mitochondrially encoded genes: 12 S and 16 S ribosomal RNAs and a group of mRNAs including cytochrome oxidase subunits I and II (COI-COII), cytochrome b (cyt b), adenosine triphosphatase (ATPase) subunits 6 and 8, and subunit 1 of the respiratory chain NADH dehydrogenase (ND1). Comparison of total testicular RNA preparations from prepuberal (6, 8, 12, 16, 18, 20, 22, and 30 days old) and sexually mature (45 days old) mice revealed no major qualitative or quantitative differences in the levels of the mitochondrial transcripts described above. Similar results were observed from enriched preparations of type A and B spermatogonia and interstitial cells obtained from the testes of 8-day-old mice. Transcripts for COI-COII, ATPase 6, or ND1 were reduced in amount in the enriched preparations of pachytene spermatocytes, round spermatids, and residual bodies when compared to the amount in total testis or liver RNA. Transcripts of all the mitochondrial genes analyzed were present in RNA preparations isolated from sperm midpiece tails obtained after sonication of epididymal spermatozoa. These studies demonstrate that (a) during testicular development the levels of mitochondrial RNA in total testicular extracts show no major qualitative and quantitative differences; (b) the mitochondrial transcripts in enriched populations of type A and type B spermatogonia are not different from those obtained from total testes extracts; (c) mitochondrial transcript levels gradually decrease in enriched preparations of pachytene spermatocytes, round spermatids, and residual bodies; and (d) the mitochondrial rRNAs and mRNAs encoded by several mitochondrial genes can be isolated from sperm midpiece tails.

Specific mRNAs in Sertoli and germinal cells of testes from stage synchronized rats.

A treatment which used vitamin A depletion followed by vitamin A repletion was used to synchronize seminiferous tubules to a few related stages of the cycle of the seminiferous epithelium. The success of the synchronization procedure was dependent on the age and size of the rat at the initiation of the experiment (20 days of age and 35-40 g) and the extent to which the vitamin A deficiency had progressed. Administration of retinol was done when the only viable germinal cells in the testis were preleptotene spermatocytes and type A spermatogonia but if the deficiency was prolonged spermatogenesis did not recover. Once established synchrony appeared to be sustained at least through several consecutive cycles. A combination of molecular probes was used to determine if the synchronized testes displayed stage specific variations in Sertoli cell and germinal cell mRNA levels as has been reported for normal asynchronized rats. Sertoli cells in the synchronized testes were shown by quantitative in situ hybridization and by Northern blot analysis to have stage specific variations in the levels of mRNA for transferrin, sulfated glycoprotein-1, and sulfated glycoprotein-2. The mRNA levels in the different stages were qualitatively similar to those in equivalent stages previously reported for testes from asynchronous rats. The germinal cell content of the synchronized testes were examined with Northern blots probed with nick-translated protamine 1 and transition protein 1 cDNAs.(ABSTRACT TRUNCATED AT 250 WORDS)