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To analyze the relationship between sperm mitochondrial membrane potential and sperm motility parameters by means of a computer-assisted sperm analyzer (CASA) and in-vitro fertilization rate(%FR).Semen samples were obtained from 26 men undergoing in vitro fertilization-embryo transfer (IVF-ET). Informed consent was obtained from all men prior to the study. Samples were prepared using wash and swim-up method in HEPES-HTF medium. The sperm motility (%MOT), progressive motility (%PMOT), average path velocity (VAP) microm/s), straight line velocity (VSL) (micro m/s), curvilinear velocity (VCL) (microm/s) and %hyperactivated sperm (%HA), and the %FR were assessed. The samples were incubated in the presence of 2.0 mciromol/L of 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) for 30 min at 37 degrees C in air and washed in PBS before flow cytometry (FACSCalibur: Becton Dickinson) analysis. The mitochondrial probe JC-1 was used to identify the mitochondrial membrane potential. The sperm was divided into three populations according to the fluorescence pattern as follows: the high mitochondrial membrane potential group (n=8), the moderate group (n=5), and the low group (n=13). Statistical analysis was performed using unpaired t-test.Significant differences were found between the high and the low groups in %MOT (91.1+/-8.5 vs 63.0+/-32.7, mean+/-SD), VAP (73.0+/-14.2 vs 52.1+/-12.5), VCL (127.0+/-28.1 vs 87.0+/-22.6), %HA (27.3+/-23.6 vs 7.2+/-9.0) and %FR [73.2 (48/56) vs 59.0 (69/117)]. No significant differences were found in other CASA parameters.When the sperm mitochondrial membrane potential increases, sperm motility parameters and fertility potential will also increase. The JC-1 dye method is useful to predict sperm fertility potential.

Recently, we have cloned the novel isoform of the progesterone receptor (PR) cDNA (PR isoform S cDNA) from the human testicular cDNA library. The isoform S cDNA consists of the novel exon (termed the exon S of the PR gene) and the exons 4-8 of the PR gene. In order to investigate the existence of the other isoform of the human PR cDNA, the human testicular cDNA library was screened by the exons 4-8 corresponding sequence of the human PR cDNA in the present study. As a result, we have identified a novel isoform of the PR cDNA (termed the PR isoform T cDNA (PR-T cDNA)), which consisted of a previously unidentified 5'-sequence and the exons 4-8 of the PR gene. The structure of this isoform T cDNA is essentially similar to that of the isoform S cDNA. By the genomic cloning, the 5'-sequence of the PR isoform T mRNA was demonstrated to originate from a novel independent exon, exon T, which was located in the 5'-upstream region of the exon S.

We have recently isolated two untranslated first exons, exon 0N and exon 0S, of rat estrogen receptor alpha (ER alpha) gene from the liver by use of 5'-rapid amplification of the cDNA ends (5'-RACE) method. In this communication, we further analyzed the 5'-untranslated region (UTR) of the ER alpha mRNA in rat anterior hypophysis in order to investigate the existence of the other 5'-untranslated exon(s) of rat ER alpha gene. Total RNA from the anterior hypophysis of 8-week-old female Wistar strain male rats was subjected to 5'-RACE with antisense primers located in exon 1 of the rat ER alpha gene and one of the positive clones (clone 35) was sequenced. The nucleotide sequence of clone 35 revealed the insertion of a previously unidentified exon (which we termed "exon 0T") between exon 0 (the first reported 5'-UTR form of rat ER alpha mRNA) and exon 1 of rat ER alpha mRNA. Analysis of rat genomic DNA indicated that exon 0T was located between exon 0 and exon 1 of rat ER alpha gene. We further investigated the distribution of ER alpha mRNA containing exon 0T in several brain regions and various peripheral tissues of 8-week-old male and female Wistar strain rats by use of reverse transcription-polymerase chain reaction (RT-PCR). The distribution of the ER alpha mRNA (0T-1) was essentially similar to that of ER alpha mRNA in which exon 0 was spliced onto exon 1 reported previously. These results indicate that (1) exon 0T is a novel untranslated exon of rat ER alpha gene which is located between exon 0 and exon 1 on rat genomic DNA, (2) exon 0T is inserted between exon 0 and exon 1 of ER alpha mRNA by alternative splicing, and (3) this alternative splicing may occur in tissues where the transcription of ER alpha gene is initiated from exon 0.

In order to investigate the regulatory mechanism of the expression of the human estrogen receptor beta (ER beta) gene, we have analyzed the structure of the 5'-untranslated region of the ER beta mRNA in the normal uterine endometrium and liver using the 5'-rapid amplification of the cDNA ends method. The sequence analysis revealed the presence of the two isoforms of the ER beta mRNA containing the distinct 5'-untranslated regions. The genomic analysis revealed that the two isoforms of the message originated from the two distinct untranslated first exons, termed the exon 0K and exon 0N, which were spliced to the exon 1. We termed the two isoforms of the message the ER beta mRNA (0K-1) and ER beta mRNA (0N-1). We further analyzed the distribution of the ER beta mRNA (0K-1) and ER beta mRNA (0N-1) in the ejaculated spermatozoa, liver, uterine endometrium and myometrium, and peripheral leukocytes using the reverse transcription-polymerase chain reaction. The distributions of the two mRNA isoforms were different from each other. From these results, it is indicated for the first time that the expression of the human estrogen receptor beta (ER beta) gene is regulated, at least in part, by the multiple untranslated first exons and promoters system.

A novel isoform (termed isoform S) of the progesterone receptor (PR) cDNA (PR isoform S cDNA) which consists of a previously unidentified 5’ sequence and exons 4–8 of the intracellular PR gene has been cloned from the human testicular cDNA library. The 5′ sequence of the message was confirmed to be derived from a novel exon (termed exon S) by genomic cloning. The expression level of the PR isoform S mRNA was higher in the spermatozoon than in the uterine endometrium with a lower expression level of the PR isoforms B and A mRNAs in the spermatozoon than in the endometrium. These results implied that the PR isoform S which was possibly translated from the PR isoform S mRNA in the spermatozoon might be related to the cell surface membrane PR. Moreover, the PR isoform S in the uterine endometium might play some physiological and/or pathogenic roles.

Recent studies on the human oestrogen receptor (ER) gene have revealed the complex system with the multiple untranslated first exons and promoters in the ER gene expression. Little information is however available on the system in the ER gene of the rat or nonhuman primate. The rat genomic library was first screened by the rat ER cDNA (0-1) probe. One of the four positive clones (lambda rEgE1) was subcloned and sequenced. The nucleotide sequence was found to contain the exon 0, the intron 0, and the exon 1 with its 3'-ends. The novel untranslated first exons, the exon ON and the exon OS, were further identified. These results indicated the presence of at least four subtypes of the rat ER mRNAs; the messages transcribed from promoter P-0 (ER mRNA (0-1)), putative promoter P-1 (ER mRNA (1-1)), promoter P-ON (ER mRNA (ON-1)) and promoter P-OS (ER mRNA (OS-1)). The P-O- or P-1 driven message (0-1) or (1-1) appeared to be expressed most strongly in major oestrogen central- (anterior pituitary, AP, hypothalamus-preoptic area, HPOA, and amygdala, AMG) and peripheral targets (uterus and ovary). The message (ON-1) was strongly expressed in the liver and kidney, but not in the HPOA, AMG, cerebral cortex, CC, and cerebellum, Ce. The OS-1 message was expressed variably but generally in the tissues examined except for the CC and Ce. Thus, the region- and tissue specific expression of the rat ER gene is likely to be regulated by the multiple untranslated exons and promoters system. Furthermore, when the ER mRNA subtypes were examined in the rat neonatal CC where the ER protein level rose transiently, considered as a model for the development of the ER or progestin receptor A and B isoforms, the expression of the ER mRNAs seemed to be differential postnatally, implicating some stage dependent usage of the promoters in the development. In the monkey, we identified the untranslated first exon OS, the homologue of the rat exon OS. Interestingly, the exon C was found to consist of two different exons, the exon OK and the exon OG. By the alternative usage of the promoters and the alternative splicing, at least six ER mRNA subtypes, that is, ER mRNAs (0-1), (1-1), (OS-1), (OS-OG-1), (OK-1) and (OK-OG-1) were identified in the monkey tissues. These messages were also differentially distributed in the monkey brain and other tissues. It was noteworthy that the P-OK driven messages were expressed almost exclusively in the monkey liver. These results have suggested that the systems of the multiple untranslated first exons and promoters and the alternative splicing are involved in the regulation of the region- and tissue specific expression of the ER gene in the brain and peripheral tissues of the rat and monkey. Stage-related usage of the promoters was also suggested in the ER gene expression in the CC of the postnatal rat in development.

In order to elucidate the mechanism of the region-specific expression of rat brain aromatase, we investigated the multiple promoter systems in the brain. Total RNA extracted from the amygdala (AMY) was subjected to a reverse transcription-polymerase chain reaction (RT-PCR) with the primers for mouse brain-specific exon I (exon I-f). The nucleotide sequence of the RT-PCR product had 89.4% homology to the corresponding region of exon I-f of the human aromatase cDNA. It was first demonstrated that aromatase mRNA with exon I-f was expressed in the rat brain. This type message was expressed in the hypothalamus-preoptic area (HPOA) but not in the cerebral cortex (CC). Furthermore, total RNAs from the HPOA, AMY and CC of the rat brain were analysed by RT-PCR with the primers for rat ovarian type message-specific exon II. Significant levels of the products were generated in the HPOA and AMY with no signals in the CC. Cortical aromatase mRNA seemed to be unique in that the tissue had no exon I-f and ovarian type mRNAs. We thus analysed the 5′-region of aromatase mRNA in the rat CC by 5′-rapid amplification of cDNA ends (RACE) with antisense primers for exon V. The upstream sequence from the 5′-end of exon IV of the aromatase RACE clones was completely different from that of the aromatase mRNA which encoded the full-translated region. These results indicated that the aromatase gene in the rat brain contained at least three promoters: hypothalamus type (exon I-f), ovarian type and cortical type, and that the region-specific expression of the brain aromatase seemed to be regulated, at least in part, by differential promoter usage.

Almost all cases with malignant transformation of a mature cystic teratoma of the ovary have been reported to be associated with squamous cell carcinoma. We describe a very rare case of mucinous cystadenocarcinoma with sarcoma-like mural nodules arising in an ovarian teratoma.

The 5′-untranslated region (UTR) of the estrogen receptor (ER) mRNA in the rat liver was analyzed by the use of the 5′-rapid amplification of the cDNA ends (5′-RACE) method. The nucleotide sequence of one of the positive RACE clones (clone 9) revealed that the existence of the novel untranslated first exon (termed “exon 0N”) being spliced onto the exon 1 of the rat ER mRNA. We further analyzed the distribution of the ER mRNA containing the “exon 0N” (ER mRNA (0N-1)) and the ER mRNA containing the previously reported exon 0 (ER mRNA (0-1)) in the rat brain and peripheral tissues. In contrast to the wide distribution of the ER mRNA (0-1), the distribution of the ER mRNA (0N-1) was almost limited in the peripheral tissues. These results indicate that the “exon 0N” is the novel untranslated first exon of the rat ER gene, and the tissue specific expression of the ER is regulated, at least in part, by differential promoter usage in the rat.

Tissue-specific expression of aromatase activity and mRNA occurs by alternative utilization of multiple untranslated first exons and promoters in the human. The major transcript in the human brain contains the brain-specific first exon, “exon I-f”. However, few reports on the untranslated first exon of aromatase mRNA in the rat brain have been available so far. In the present study, we investigated the existence and expression of exon I-f in the rat brain to elucidate the mechanism of the tissue-specific expression of the brain aromatase. Total RNA extracted from amygdala (AMY) was subjected to a reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence of the RT-PCR product had 89.4% homology to the corresponding region of exon I-f of the human aromatase cDNA. It was indicated that the major transcript in the rat AMY contained exon I-f by the use of a rapid amplification of cDNA ends (RACE). Furthermore, in order to determine the distribution of the aromatase mRNA with exon I-f, total RNAs from the hypothalamus-preoptic area (HPOA), AMY, testis and ovary were analysed by RT-PCR using the primers specific for the mouse exon I-f and the primers for the rat exon III-V. Significant levels of PCR products were found in all tissues with the highest level being in the ovary, using the primers for exon III-V. On the other hand, using the primers for exon I-f, the levels of signals from HPOA and AMY were higher than those from the testis and ovary. These results suggest that tissue-specific expression of aromatase mRNA occurs by an alternative utilization of multiple promoters in the rat, as in the human. It should be noted that minor transcripts containing exon I-f were observed in the testis and ovary.

To elucidate the mechanism of the region-specific expression of the aromatase in the primate brain, we investigated the distribution and level of the total aromatase mRNA and the aromatase mRNA with the exon 1-f, which was reported to be the brain-specific exon 1 of the human aromatase gene, in male Japanese monkeys. Total RNAs extracted from the hypothalamus-preoptic area (HPOA), amygdala (AMY), cerebellum, hippocampus, brainstem, five regions of cerebral cortex and four peripheral tissues: liver, kidney, adipose tissue and testis were subjected to a semi-quantitative reverse transcription-polymerase chain reaction-Southern blotting (RT-PCR-SB) assay. The levels of the total aromatase mRNA was high in the HPOA, AMY and testis with a low level of message in the other regions. These results roughly paralleled the distribution of aromatase activity of the monkey brain previously reported. The level of the aromatase mRNA with the exon 1-f was high in the HPOA and AMY, and low in the other regions of the brain and the testis with an undetectable level of the messenger in the other peripheral tissues. Furthermore, the ratio of the aromatase mRNA with the exon 1-f to the total aromatase mRNA was different among various regions of the monkey brain, for example, the ratio in the AMY was distinctly higher than that in the HPOA. These results indicated that the level of the aromatase mRNA mainly regulated the level of aromatase protein and aromatase activity in a region-specific manner, and that the exon 1-f was used in most of the monkey brain regions. Moreover, the ratio of the aromatase mRNA with the exon 1-f to the total aromatase mRNA varied in the brain regions. It was implied that the aromatase mRNA using the other first exons was also expressed in the brain and was involved in the region-specific expression of the brain aromatase.

Progesterone receptors (PRs) are known to exist in two forms: a larger molecular, form B, and a smaller one, form A. Rat PR cDNA corresponding to the region around the translation-initiation site (ATGB) of the putative PR form B mRNA was cloned, together with cloning of steroid-binding domain of the PR forms A and B. An imperfect "estrogen responsive element," GGTCG*** TGACT, was located around ATGB of the rat PR cDNA. The distribution of PR mRNA-containing neurons was mapped in the female adult rat brain by in situ hybridization, which was largely in agreement with that of PR proteins. Differential intracerebral distribution of the mRNAs, measured by the quantitative RT-PCR Southern blotting assay, was found between the total (A+B) and form B mRNA levels, indicating possible distinct mechanisms responsible for regulation of the expression of the PR mRNAs. A region-specific and stage-related gene expression of form B seems predominantly to be "turned on" first around birth, followed by that of form A around Days 8-12. The postnatal developmental pattern of PR form B mRNA in the cerebral cortex resembled that of the PR proteins. Noninducibility of the cortical PR by estrogen might be ascribable to the predominance of form B mRNA, which was reported to have little or no estrogen inducibility. A missmatch existed between PR from A and its mRNA levels, suggesting some impairment of the synthesis of form A. Thyroid hormone may be a regulator of gene expression of the receptor in the developing cortex.

The effects of neonatal hypothyroidism on the gene expression of progesterone receptors (PRs) in the 8-day-old female rat brain were examined. The levels of PR ('A'+'B')-, PR ('B')-, estrogen receptor (ER)- and beta-actin mRNAs in the cerebral cortex and hypothalamus-preoptic area of propylthiouracil-treated rats (PTU group) or untreated rats (control group) were analyzed using a quantitative reverse transcription-polymerase chain reaction-Southern blotting assay. When the levels of PR mRNAs were calibrated by respective levels of beta-actin mRNA, the levels of PR ('A'+'B')- and PR ('B') mRNAs in the cerebral cortex of the PTU group were markedly less than those in the control group with no significant changes in the levels of PR messages in the hypothalamus-preoptic area of both groups. No significant difference in the calibrated levels of ER mRNA between both groups was found in these tissues. These results on the PR-, and ER mRNAs were essentially similar to those on the levels of PR- and ER proteins previously reported suggesting that thyroid hormones affect the transcriptional machinery of PR in the developing brain, in a region-specific manner.

Distribution and postnatal changes of aromatase mRNA in the Wistar strain female rats brain were investigated to elucidate the mechanism of the region-specific and stage-related regulations of brain aromatase activity. Total RNAs extracted from the hypothalamus-preoptic area (HPOA), amygdala, cerebral cortex, cerebellum and anterior hypophysis were subjected to a quantitative reverse transcription-polymerase chain reaction-Southern blotting (RT-PCR-SB) assay. The levels of aromatase mRNA were as follows: amygdala > cerebral cortex not equal to HPOA >> cerebellum not equal to anterior hypophysis. These results roughly paralleled the distribution of the aromatase activity and the aromatase protein reported previously, with the exception of that of the cerebral cortex. The postnatal developmental patterns of aromatase mRNA in the HPOA and cerebral cortex were further studied. The levels of aromatase mRNA in the HPOA tended to be high around birth (day 0) and to decrease at days 4-8 followed by no remarkable change to an adult. The developmental pattern in the HPOA was essentially similar to that of aromatase activity. On the other hand, in the cerebral cortex where very low or no aromatase activity was reported throughout developmental stages, aromatase mRNA was low but detectable level around day 0, thereafter gradually increased to an adult level. From these results, it is inferred that synthesis of the aromatase protein by the level of the mRNA seems to mainly regulate the aromatase activity in a region-specific and stage-related fashion in most parts of the rat brain, except for the cerebral cortex where the posttranscriptional regulation may play an important role.

The postnatal development of the progestin receptor (PR) system in the rat brain is a region-specific and stage-related process. In an attempt to analyze the molecular mechanism by which the dramatic change of gene expression of the PR occurs we have examined the level of PR mRNAs in the hypothalamus-preoptic area (HPOA) and cerebral cortex in development from fetal to postnatal stages of female rats. We used polymerase chain reaction to clone, from uterine cDNA, the cDNA corresponding to the steroid-binding domain of the PR forms 'A' and 'B' mRNA as well as the region around the translation-initiation site (ATG1) of the putative PR form 'B' mRNA. A quantitative reverse transcription-polymerase chain reaction assay was used to measure the level of mRNAs for PR forms 'A' and 'B' (total PR mRNAs) and PR form 'B'. There was a regional difference in the intracerebral distribution between the total and form 'B' mRNAs, indicating possible distinct mechanisms responsible for regulating the expression of the PR mRNAs. The PR mRNAs in the brain, already detectable 2 days before birth, increased at early neonatal stages. The total PR mRNAs in the cortex developed in a manner essentially similar to the PR protein at the early stages, but, surprisingly, unlike the receptor, the messages remained high at the later stages from day 18 to 8 weeks of life. On the other hand, the ontogeny of the cortical mRNA for form 'B', which predominantly existed in the region, resembled that of the cortical PR protein. In the HPOA the postnatal development of the form 'B' mRNAs was also roughly similar to the PR. These results suggest region-specific and stage-related gene expression of the PR isoform system in the developing brain: gene expression of form 'B' seems to be predominantly, first, "turned on" around birth, followed by form 'A' mRNA expression around days 8-12. Moreover, lowered levels of the cortical PR mRNAs in the propylthiouracil-induced hypothyroid rat, together with suppressed PR level, indicate a possible regulatory role of thyroid hormone on gene expression of the cortical receptor.

In order to examine the existence and level of androgen receptor messenger ribonucleic acid (ARmRNA) in the rat brain, reverse transcription-polymerase chain reaction (RT-PCR)-Southern blot analysis was carried out. Total RNA extracted from each tissue was reverse transcribed, followed by PCR with two oligonucleotide primers specific for a part (458 bp) of the androgen binding domain of the rat ARcDNA. It was confirmed by direct nucleotide sequencing that the amplified fragment corresponded to part of the rat ARcDNA. To detect and quantify the amplified fragments, a Southern blot analysis was carried out. The levels of amplified fragments were calculated from the standard curve obtained from graded diluted adrenal total RNAs. In the present study, it was revealed that the RT-PCR-Southern blot analysis possessed a high-degree of sensitivity and allowed the quantitative estimation of mRNA. With this method, amplified fragments were obtained from all five brain regions examined. The results indicate that ARmRNA is widely distributed in the whole brain. Moreover, since the ARmRNA level roughly paralleled the AR protein level, it seems that the AR protein level in the brain may be primarily regulated by the ARmRNA level.

Our previous reports have revealed the postnatal developmental pattern of progesterone receptor (PR) in the rat cerebral cortex, but little is known about PRmRNA changes in the tissue so far. In the present study, we have attempted to detect and quantify PRmRNA in the tissue by the use of the reverse transcription-polymerase chain reaction (RT-PCR)-dot blot analysis. Cerebral cortical tissues were dissected from female Wistar rats at 2 and 8 days of age. Total RNA extracted from the tissues was reverse transcribed, followed by PCR. The PCR primer set, whose sequence was derived from the human clone, flanked the part (320 bp) of the human PRcDNA which corresponded to the progesterone binding domain. The 320 bp of the RT-PCR product was generated from rat uterine RNA. The amino acid sequence deduced from the nucleotide sequence of the product had a 95.5% identity with the corresponding region in human PR, confirming that the product had orginated from the rat PRmRNA. Moreover, a highly sensitive and quantitative assay for rat PRmRNA had been developed by the use of RT-PCR-dot blotting. The PRmRNA was detectable in the cerebral cortex of both 2- and 8-day-old rats by the assay. Furthermore, the level of cortical PRmRNA of the 8-day-old rats were greater than the 2-day-old rats. These results indicate that the RT-PCR assay is useful for detection and quantification of PRmRNA in the neonatal rat cerebral cortex, and that increment of the cortical PRmRNA in the 8-day-old rat is associated with a drastic change of the level of the cortical PR around day 10.

Highlights • We present an unusual case of umbilical endometriosis and huge uterine leiomyomas with marked hydropic and cystic degeneration. • Although umbilical tumors are uncommon, the differential diagnosis should include umbilical endometriosis, particularly in reproductive-age women. • Umbilical endometriosis can be suspected from the clinical presentation, but the diagnosis should be confirmed with histological examination.