Biomedical subjects

R L Stouffer

Publications and source records attributed to R L Stouffer.

At least 37 records · Page 2Linked to original sources

PubMed1999

Vascular endothelial growth factor (VEGF) and angiopoietin regulation by gonadotrophin and steroids in macaque granulosa cells during the peri-ovulatory interval.

The role of endothelial cell-specific growth factors in the vascularization of the primate peri-ovulatory follicle was examined. Experiments were designed firstly to detect expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in granulosa cells and secondly, to determine whether gonadotrophins and/or steroids regulate their expression during the peri-ovulatory interval. Granulosa cells and follicular fluid were collected from rhesus macaques undergoing ovarian stimulation before (0 h), 12, or 36 h after a bolus of ovulatory human chorionic gonadotrophin (HCG), with or without steroid ablation and progestin replacement. VEGF, Ang-1 and Ang-2 mRNA were all detected prior to the ovulatory stimulus. Whereas follicular fluid VEGF concentrations increased 6-fold (P < 0.05) between 0 and 12 h, VEGF mRNA values were unchanged and were unaffected by steroid ablation. Ang-1 mRNA decreased from 0 to 12 h (P < 0.05), followed by a 30-fold increase (P < 0.05) at 36 h, while Ang-2 mRNA values were unchanged between 0, 12 and 36 h. Steroid ablation decreased (P < 0.05) Ang-1 mRNA at 36 h, and Ang-2 mRNA at 12 h, while only Ang-1 was restored by progestin replacement. These data suggest a dynamic expression of vascular-specific growth factors in a gonadotrophin-dependent, steroid-independent (VEGF) or steroid-dependent (Ang-1) manner in granulosa cells of peri-ovulatory follicles of primates.

3-Hydroxysteroid Dehydrogenases↗

PubMed1999

Expression of matrix metalloproteinases and their tissue inhibitor messenger ribonucleic acids in macaque periovulatory granulosa cells: time course and steroid regulation.

Progesterone appears essential for ovulation and luteinization of the primate follicle, but specific gene targets of progesterone action remain elusive. Limited evidence supports a role for progesterone in the induction of collagenolytic activity in the periovulatory follicle of primate and nonprimate species. This study was designed to elucidate the pattern of expression and progesterone regulation of mRNAs for the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in macaque granulosa cells during controlled ovarian stimulation cycles before (0 h) and after (up to 36 h) administration of an ovulatory hCG bolus. Levels of mRNAs for interstitial collagenase, gelatinase A, matrilysin, TIMP-1 and TIMP-2 increased (p < 0.05) within 12 h of hCG, while gelatinase B mRNA increased later, by 36 h after hCG. Administration of a 3beta-hydroxysteroid dehydrogenase inhibitor (Trilostane [TRL]) during hCG treatment decreased (p < 0.05) mRNA levels for interstitial collagenase, gelatinase B, matrilysin, TIMP-1, and TIMP-2. Progestin (R5020) replacement during hCG+TRL treatment returned interstitial collagenase and TIMP-1 mRNAs to control levels. These data suggest that one action of progesterone, and possibly other steroids, in the cascade of events leading to ovulation and luteinization of the primate follicle is to regulate the expression of specific ovarian proteases and protease inhibitors.

3-Hydroxysteroid Dehydrogenases↗

PubMed1999

Gonadotropin and steroid regulation of steroid receptor and aryl hydrocarbon receptor messenger ribonucleic acid in macaque granulosa cells during the periovulatory interval.

Although steroids play a local role(s) in ovulation and luteinization of the primate follicle, the dynamics of steroid receptor expression during the 36- to 38-h periovulatory interval has yet to be elucidated. The present study examines the regulation of messenger RNAs (mRNAs) for progesterone (PR), androgen (AR), and estrogen (ER alpha, ER beta) receptors as well as the aryl hydrocarbon receptor (AhR) in macaque granulosa cells during controlled ovarian stimulation cycles before (0 h) and after (up to 36 h) administration of the ovulatory hCG bolus with or without steroid depletion and progestin replacement. All steroid receptor mRNAs were detected in granulosa cells before the ovulatory stimulus, as determined by RT-PCR. PR mRNA increased (P < 0.05) by 12 h after hCG; 24 and 36 h after hCG, levels were intermediate between 0-12 h. PR mRNA was reduced by steroid depletion throughout the periovulatory interval (P < 0.05); however, progestin replacement returned PR mRNA to control levels at 12 h. AR mRNA increased (P < 0.05) at 24 h post-hCG and remained at this level 36 h after hCG; steroid depletion did not alter AR mRNA levels. ER alpha mRNA did not change, whereas ER beta decreased 12-36 h after the ovulatory stimulus (P < 0.05). Steroid depletion reduced ER alpha mRNA 12 h after hCG, an effect partially reversible by progestin replacement, whereas ER beta mRNA was not affected by steroids. AhR mRNA was undetectable before the administration of hCG, but increased by 12 h (P < 0.05). These data demonstrate hCG-initiated, steroid-dependent (PR, ER alpha) and -independent (AR, ER beta, AhR) expression of receptor mRNAs in primate granulosa cells during the periovulatory interval. Differences in patterns of expression may relate to diverse roles for steroid hormones and AhR ligands in periovulatory events.

Animals↗

PubMed1999

Titrating luteinizing hormone replacement to sustain the structure and function of the corpus luteum after gonadotropin-releasing hormone antagonist treatment in rhesus monkeys.

These studies were designed to identify 1) a regimen of a third generation GnRH antagonist that abolishes primate luteal function, and 2) the amount of LH replacement required to maintain the structure and functional life span of the corpus luteum of the menstrual cycle after GnRH antagonist treatment. A single injection of antide at 3 or 5 mg/kg BW on day 6 of the luteal phase suppressed serum progesterone levels within 1 day of treatment, but levels recovered within 4 days. Administration of antide (3 mg/kg) for 3 days (luteal days 6-8) reduced (P < 0.05) serum progesterone below 1 ng/mL and maintained these low levels for the entire sampling period; in subsequent experiments, all monkeys received this antide regimen. Fixed doses (5, 10, or 20 IU) of recombinant human LH administered at 8-h intervals during and after antide treatment stimulated progesterone production in a dose-dependent manner; these monkeys menstruated earlier than controls regardless of treatment group. Replacement with an escalating dose regimen (5-20 IU) of LH resulted in typical serum progesterone and relaxin levels throughout a luteal phase of normal length. Corpora lutea removed on day 10 from monkeys treated with antide alone had decreased wet weight (P < 0.05) and few large luteal cells; coadministration of the escalating dose regimen of LH maintained luteal structure similar to that seen in time-matched controls. Antide-only treatment increased progesterone receptor (PR) messenger ribonucleic acid, but decreased PR immunostaining in luteal tissue; the escalating dose regimen of LH maintained PR messenger ribonucleic acid and immunostaining similar to those in controls. This study indicates that during GnRH antagonist administration, an escalating dose regimen of LH replacement is optimal for maintenance of the structure and functional life span of the primate corpus luteum.

Animals↗

PubMed1999

Androgen receptor mRNA expression in the rhesus monkey ovary.

Immunocytochemical detection of androgen receptors (ARs) in several compartments of the macaque ovary, including the germinal epithelium, follicle, and corpus luteum, suggests a role for androgens in modulating ovarian function via the classical receptor-mediated pathway. To examine AR mRNA expression in the rhesus monkey ovary, total RNA was isolated from whole ovaries, the germinal epithelium-enriched cortical and medullary compartments of the ovary, and corpora lutea from early (d 3-5), mid (d 6-8), mid-late (d 10-12), and late (d 13-15) stages of the luteal phase of the menstrual cycle. RNA was also obtained from luteinized granulosa cells from monkeys receiving gonadotropin treatment to stimulate the development of multiple ovarian follicles. After reverse transcription of total RNA using oligo-dT as a primer, polymerase chain reaction (PCR) was used to amplify a unique 329 bp segment of the monkey AR hormone-binding region. Reverse transcriptase (RT)-PCR products of the expected size were detected in all ovarian and control tissues. Sequence analysis of the AR cDNA from the macaque ovary revealed 99% nucleotide homology and 100% predicted amino acid homology to the cDNA for the hormone-binding region of human AR. Northern analysis demonstrated the presence of a major AR mRNA species at 9.5 kb in corpus luteum, luteinized granulosa cells, and prostate, with additional bands detected in the corpus luteum and prostate at 7.9 and 3.4 kb, respectively. A sensitive RNase protection assay was used to examine AR mRNA levels in ovarian tissues and showed AR mRNA expression throughout the life-span of the corpus luteum. Thus, detection of AR mRNA in the primate ovary, including the periovulatory follicle and corpus luteum, supports the concept that these tissues are targets for receptor-mediated androgen action during the menstrual cycle.

Animals↗

PubMed1998

Recent progress in mammalian cloning.

PURPOSE: Our purpose was to review recent progress in the use of nuclear transfer technology to produce genetically identical mammals. METHODS: A literature review was conducted. RESULTS: The reasons for cloning nonhuman mammals are manyfold including commercial, biomedical, and basic research applications. Individual steps in the nuclear transfer process are itemized, along with a detailed description of the specific approaches used in the production of Dolly, NETI, and DITTO. The potential application of nuclear transfer in the treatment of human infertility is also considered, along with bioethical concerns. Finally, insights are provided concerning the future application of cloning technology in rhesus macaques. CONCLUSIONS: The cloning of a lamb (Dolly) from an adult, mammary gland cell coupled with the successful production of rhesus monkeys (NETI and DITTO) by nuclear transfer of embryonic cells marks the beginning of a "Golden Age" in the development and application of somatic cell cloning technology in mammals.

Animals↗

PubMed1998

Chronic treatment of female rhesus monkeys with low doses of the antiprogestin ZK 137 316: establishment of a regimen that permits normal menstrual cyclicity.

Large doses of antiprogestin typically disrupt menstrual cyclicity. A chronic low-dose regimen of the potent new antiprogestin ZK 137 316, which permits continued menstrual cyclicity but alters gonadal-reproductive tract activity, was established. Rhesus monkeys received vehicle (n = 6) or 0.01 (n = 8), 0.03 (n = 8) or 0.1 (n = 5) mg ZK 137 316/kg body weight daily for five menstrual cycles (C-1 to C-5). Oestradiol, progesterone and gonadotrophin profiles were normal during cycles involving vehicle and 0.01 and 0.03 mg ZK 137 316/kg body weight. In the 0.1 mg/kg group, mid-cycle oestradiol and gonadotrophin surges, and subsequent progesterone production, were absent in C-3 and C-5. Ovarian cyclicity was accompanied by timely menstruation in the vehicle and 0.01 mg/kg groups. By C-3, half the animals in the 0.03 mg/kg group and all animals in the 0.1 mg/kg group were amenorrhoeic. A corpus luteum was noted during the mid-luteal phase of C-5 in the vehicle, 0.01 mg/kg and 0.03 mg/kg groups. Large antral and cystic follicles were evident in the 0.1 mg/kg group. Thus, a daily treatment with 0.01 mg/kg ZK 136317 permitted normal menstrual cyclicity in macaques. While the daily administration of 0.03 mg/kg ZK 136 317 allowed ovarian cyclicity, menstruation was disrupted in some animals. Increasing the dose to 0.1 mg/kg antagonized pituitary function and resulted in anovulation and amenorrhoea. A chronic low-dose regimen of the antiprogestin ZK 137 316, which permits normal ovarian/menstrual cyclicity, has potential as a contraceptive in women.

Animals↗

PubMed1998

Chronic treatment of cycling rhesus monkeys with low doses of the antiprogestin ZK 137 316: morphometric assessment of the uterus and oviduct.

The long-term effects of the antiprogestin ZK 137 316 on reproductive tract morphology in rhesus macaques were investigated. The monkeys were injected daily (i.m.) for five menstrual cycles with vehicle or 0.01, 0.03 or 0.1 mg ZK 137 316/kg body weight. Reproductive tracts (n = 3/ group) were collected during the mid-luteal phase (day 8) of the fifth cycle in the control, 0.01 and 0.03 mg/kg groups, or 6-7 days after the oestradiol peak in the 0.1 mg/kg group. ZK 137 316 treatment resulted in a dose-dependent atrophy of the endometrium, marked by reduced mitotic activity in the glands, compaction of the stroma, degradation of spiral arteries and dilation of veins. There was no effect of ZK 137 316 on myometrial or oviductal weight. Treatment with 0.1 and 0.03 mg/kg, but not 0.01 mg/kg resulted in fully ciliated and secretory oviducts, indicating a dose-dependent blockade of progesterone antagonism of oestrogen-dependent oviductal differentiation. In the endometrium, the suppressive action of progesterone on oestrogen and progestin receptors was also blocked by ZK 137 316 in a dose-dependent manner. However, endometrial atrophy appeared due to inhibition of progesterone action together with a blockade of oestrogen-dependent proliferation. The profoundly suppressed endometrium produced by chronic low-dose ZK 137 316 treatment is unlikely to support implantation. Such treatment may therefore provide a novel contraceptive modality.

Animals↗

PubMed1998

A bolus of recombinant human follicle stimulating hormone at midcycle induces periovulatory events following multiple follicular development in macaques.

The efficacy of follicle stimulating hormone (FSH) as an alternative to luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) for the initiation of periovulatory events in primate follicles is unknown. A single bolus of 2500 IU recombinant (r)-hFSH was compared to 1000 IU r-HCG for its ability to promote oocyte nuclear maturation and fertilization, granulosa cell luteinization and corpus luteum function following r-hFSH (60 IU/day) induction of multiple follicular development in rhesus monkeys. Following the r-hFSH bolus, bioactive luteinizing hormone concentrations were <3 ng/ml. Peak concentrations of serum FSH (1455+/-314 mIU/ml; mean+/-SEM) were attained 2-8 h after r-hFSH, and declined by 96 h. Bioactive HCG concentrations peaked between 2-8 h after r-HCG and remained > or = 100 ng/ml for >48 h, while immunoreactive FSH concentrations were at baseline. The proportion of oocytes resuming meiosis and undergoing in-vitro fertilization (IVF) were comparable for r-hFSH (89%; 47+/-19%) and r-HCG (88%; 50+/-17%). In-vitro progesterone production and expression of progesterone receptors in granulosa cells did not differ between groups. Peak concentrations of serum progesterone in the luteal phase were similar, but were lower 6-9 days post-FSH relative to HCG. Thus, a bolus of r-hFSH was equivalent to r-HCG for the reinitiation of oocyte meiosis, fertilization and granulosa cell luteinization, but a midcycle FSH surge did not sustain normal luteal function in primates.

Animals↗

PubMed1998

A chronic, low-dose regimen of the antiprogestin ZK 137 316 prevents pregnancy in rhesus monkeys.

Continual administration of low doses of the antiprogestin ZK 137 316 was previously reported to permit ovarian/menstrual cyclicity, but disrupt endometrial growth in macaques. The contraceptive efficacy of this regimen was tested in female rhesus monkeys (10 per group) treated daily with vehicle (controls), 0.01 or 0.03 mg ZK 137 316 per kg body weight for 30 days before and during continual co-habitation with males of proven fertility. Treatment continued until confirmation of pregnancy or for 5 months after pair-housing with males. Mating and vaginal sperm were evident in all females. A cumulative pregnancy rate of 90% (9/10) was observed in the controls. Of the 10 animals receiving 0.01 mg/kg, four conceived during the first 2 months of pairing (P = 0.06) with no further conceptions. No pregnancies were observed in the 0.03 mg/kg group (P < 0.01). Timely, overt menses occurred at a higher frequency in the 0.01 mg/kg group than the 0.03 mg/kg group. However, corpora lutea were present in ovaries from both groups during the last treatment cycle, indicating that ovarian cycles occurred. Thus, chronic administration of low-dose ZK 137 316 that permits continued ovarian cyclicity and a high incidence of timely menses, prevents pregnancy in non-human primates. This regimen may provide a novel method of contraception for women.

Animals↗

PubMed1997

Vascular endothelial growth factor levels in serum and follicular fluid of patients undergoing in vitro fertilization.

OBJECTIVE: To define the relationship between serum and follicular fluid (FF) levels of vascular endothelial growth factor (VEGF), E2, and P in patients undergoing IVF; to quantify the effects of hCG on serum levels of VEGF during early pregnancy, and to report serial measurements of serum and ascites fluid levels of VEGF in a patient with severe ovarian hyperstimulation syndrome (OHSS). DESIGN: Prospective observational study. SETTING: University IVF program. PATIENTS(S): Women undergoing conventional IVF, receiving donated oocytes or spontaneously conceiving. One patient hospitalized with severe OHSS. MAIN OUTCOME MEASURE(S): Concentrations of VEGF, E2, and P in serum, FF, or peritoneal fluid. RESULT(S): At the time of egg retrieval, FF VEGF concentrations were positively correlated with serum and FF P concentrations and with patient age. At 11 to 14 days after ET, pregnant recipients of autologous fresh embryos had higher serum VEGF levels than both nonpregnant recipients of autologous fresh embryos and pregnant recipients of donor eggs. Elevated serum VEGF levels in a patient with severe OHSS coincided with the clinical onset and recurrence of symptoms. CONCLUSION(S): In patients undergoing IVF, FF VEGF levels at the time of egg retrieval correlated with the degree of follicular luteinization. There is a significant ovarian contribution to circulating VEGF levels during early gestation. Elevated serum VEGF levels may be a factor in the etiology of OHSS symptoms.

Adult↗

PubMed1997

Vascular endothelial growth factor production by human luteinized granulosa cells in vitro.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) originating from the follicle or corpus luteum may be a physiological regulator of ovulation and neovascularization of luteinizing tissue, as well as a pathological factor in the development of ovarian hyperstimulation syndrome (OHSS). The objective of this study was to quantify VEGF production by human luteinized granulosa cells in vitro and to determine if gonadotrophin stimulates VEGF production directly and/or indirectly via enhanced synthesis of progesterone. In study 1, luteinized granulosa cells collected from women undergoing ovarian stimulation for in-vitro fertilization were cultured in the presence and absence of human chorionic gonadotrophin (HCG; 100 ng/ml) and/or low density lipoprotein (LDL; 100 microg protein/ml). In study 2, the progesterone synthesis inhibitor trilostane (250 ng/ml) and/or a progesterone receptor antagonist ZK137.316 (3.2 microM) were also added. Medium was harvested on days 1, 3, 5, 7 and 9 of culture and assayed for VEGF and progesterone. Results of study 1 were divided into two categories based on control concentrations of VEGF on day 1: 'low producers' (n = 6; <750 pg VEGF/ml) and 'high producers' (n = 5; >1000 pg VEGF/ml; P < 0.01). VEGF concentrations in cultures of both low and high producers increased (P < 0.01) from day 1 to maximal values on day 3, then steadily declined through to day 9. Chronic exposure to LDL or HCG increased (P < 0.05) VEGF concentrations in cultures of low producers by day 3 and day 5 respectively. In contrast, LDL did not alter VEGF concentrations in cultures of high producers and HCG did not increase VEGF concentrations until day 7. Nevertheless, acute exposure to HCG beginning on day 7 increased (P < 0.05) VEGF concentrations 3-fold in cultures of low or high producers. In study 2, trilostane treatment decreased (P < 0.05) progesterone concentrations by 91% on day 1 of culture but had no effect on VEGF concentrations on any day. ZK137.316 alone or with trilostane did not affect VEGF synthesis. These results suggest that VEGF production by luteinized granulosa cells is enhanced by gonadotrophin (HCG) independent of gonadotrophin-stimulated progesterone synthesis. These data are consistent with the hypothesis that the exacerbation of OHSS in early pregnancy is mediated by the CG stimulation of luteal VEGF production.

Adult↗

PubMed1997

Initiation of periovulatory events in gonadotrophin-stimulated macaques with varying doses of recombinant human chorionic gonadotrophin.

During in-vitro fertilization (IVF) cycles, a large bolus of human chorionic gonadotrophin (HCG) is used to induce periovulatory events, but the efficacy of lower doses is undefined. Following follicular stimulation in rhesus monkeys, oocyte nuclear maturation, IVF, granulosa cell luteinization and corpus luteum function were compared after injection of 100, 300 or 1000 IU recombinant HCG or 1000 IU urinary HCG. Bioactive HCG rose to peak concentrations within 2 h that were proportional to the dose administered (100 < 300 < 1000 IU, recombinant HCG = urinary HCG). The duration of surge values (>100 ng/ml) was also dose-dependent (0 h, 100 IU; 24 h, 300 IU; >48 h, 1000 IU, recombinant and urinary HCG). While the proportions of oocytes resuming meiosis and undergoing IVF were similar among groups, fewer animals yielded fertilizable oocytes following 100 and 300 IU (five of nine) compared to 1000 IU recombinant and urinary HCG (nine of 10). Peak values of serum progesterone in the luteal phase were similar, but declined 2 days earlier after 100 and 300 IU relative to 1000 IU recombinant and urinary HCG. Thus, 3-10 fold lower doses of HCG elicit low amplitude surges of short duration that induce periovulatory events such as re-initiation of oocyte meiosis and granulosa cell luteinization. However, oocyte fertilization and luteal function may optimally require surges of higher amplitude and longer duration similar to those produced by standard doses of 1000 IU recombinant or urinary HCG.

Animals↗

PubMed1997

Localization of steroidogenic enzymes in macaque luteal tissue during the menstrual cycle and simulated early pregnancy: immunohistochemical evidence supporting the two-cell model for estrogen production in the primate corpus luteum.

It is hypothesized that the two-cell model for estrogen production by the ovarian follicle is preserved in the primate corpus luteum, but there is little direct evidence to support this theory. To determine the sites of androgen and estrogen synthesis within the primate corpus luteum and to ascertain whether changes in steroid hormone levels are related to steroidogenic enzyme expression, the enzymes converting progesterone to androgen (cytochrome P450 17alpha-hydroxylase/17,20 lyase; P450(c17)) and then to estrogen (aromatase; P450(arom)), as well as P450 side-chain cleavage (P450(scc)) and 3beta-hydroxysteroid dehydrogenase (3beta HSD), were detected by immunohistochemistry in macaque luteal tissue throughout the menstrual cycle and simulated early pregnancy. Corpora lutea were collected from rhesus monkeys in the early (Days 2-4 post-LH surge), mid (Days 6-8), mid-late (Days 10-12), and late (Days 14-15) luteal phase and after 1, 3, 6, or 9 days of hCG treatment that began on Day 9 of the luteal phase. Specific cytoplasmic staining for P450(c17), P450(arom), P450(scc), and 3beta HSD was present in luteal cells, but not in the microvasculature, within all luteal tissues examined. P450(c17)-stained luteal cells were located along the vascular tracts and around the periphery of the corpus luteum. Intensely stained luteal cells were associated with blood vessels entering from the outer surface of the corpus luteum, but not with blood vessels returning from the connective tissue centrum. In contrast, P450(arom)-stained luteal cells were distributed throughout the luteal parenchyma. P450(c17) staining intensity was similar at all stages of the luteal phase; however, the number and intensity of P450(arom)-stained cells decreased by late luteal phase. In simulated early pregnancy, cells stained for P450(c17) were present near blood vessels, with some positive cells scattered throughout the corpus luteum. P450(arom) immunostaining was heterogeneous within the corpus luteum; many intensely stained cells were interspersed among others that were only lightly stained. Overall, cellular staining for P450(c17) and P450(arom) remained intense through 9 days of simulated early pregnancy. In contrast, P450(scc) and 3beta HSD immunoreactivity were not located in distinct luteal compartments. These results are consistent with a two-cell model for steroid hormone production in the primate corpus luteum, whereby paraluteal (theca-luteal) cells produce androgen substrate that is converted to estrogens by true (granulosa-) luteal cells. The divergence in enzyme detection as the luteal phase progresses, with P450(c17) labeling high and P450(arom) staining having decreased, suggests a shift in the function of the corpus luteum as it ages. Enzyme localization during chorionic gonadotropin exposure simulating early pregnancy demonstrates the continued capacity of the primate corpus luteum to produce steroid hormones.

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

Rhesus monkeys produced by nuclear transfer.

Genetically identical nonhuman primates can provide a powerful animal model for gene therapy and research activities where the physiological parameters directly or indirectly under study are heritable. Here we demonstrate that nuclear transfer is a viable technology for the production of identical rhesus macaques. Oocytes recovered from gonadotropin-treated females were enucleated by aspiration of the first polar body and underlying ooplasm, then activated by cycloheximide exposure. Individual diploid blastomeres, recovered from in vitro-fertilization-produced embryos (either fresh or frozen-thawed) and used as nuclear donors, were injected under the zona pellucida of enucleated (chromosome-free) oocytes and fused by electric pulses. The reconstituted embryos were cocultured on buffalo rat liver cells before cryostorage and transfer to synchronized host mothers. Of the 9 females receiving a total of 29 reconstituted embryos, 3 became pregnant, with two live births resulting, one male and one female. The parentage of both infants was established unequivocally by genotype analysis at 7 highly variable short tandem repeat loci.

Animals↗

PubMed1997

The ratio of progesterone receptor isoforms changes in the monkey corpus luteum during the luteal phase of the menstrual cycle.

Recent evidence suggests that progesterone is required for ovulation, luteinization, and the maintenance of luteal structure and function in primates. Progesterone action is mediated by intracellular progesterone receptors (PRs), and PRs are detectable by immunocytochemistry in the monkey corpus luteum. However, changes in total luteal PR and PR isoform expression have not been quantitated in the corpus luteum during its life span in the menstrual cycle. This study was initiated to identify and quantify PR isoforms in the macaque corpus luteum throughout the luteal phase of the natural cycle by means of Western blotting. Several antibodies generated against the human PR recognized two bands of consistent molecular weights in monkey tissues, and these bands comigrated with PR-A and PR-B from human T47D cells. Taken together, these data suggest that the two proteins identified were macaque PR-A and PR-B. The estimated molecular weights of monkey PR-A and PR-B were approximately 90,000 and 120,000, respectively. PRs were detected in a variety of macaque tissues, including the endometrium, whole ovary, and decidua, but not in spleen, which is PR-negative by other techniques. Whereas PR-A was the predominant isoform observed in endometrium and decidua, PR-B predominated in the ovary without a dominant follicle or corpus luteum as well as in the corpus luteum. In luteal tissue, PR-A levels decreased (p < 0.05) over the course of the luteal phase, while PR-B levels were unchanged. Hence the ratio of PR-B to PR-A (PR-B:PR-A) increased (p < 0.05) from early to very late luteal phase. Since PR-B:PR-A can alter gene expression in response to progestins and antiprogestins in vitro, the temporal changes in PR-B:PR-A in the monkey corpus luteum may contribute to functional differences in luteal responses to progesterone and other steroids in vivo.

Animals↗

PubMed1997

Gonadotropin versus steroid regulation of the corpus luteum of the rhesus monkey during simulated early pregnancy.

During the nonconceptive cycle in primates, progesterone is a likely intermediary for several LH-dependent events in the ovary including ovulation, luteinization of the follicle wall, and maintenance of the developed corpus luteum. To determine whether progesterone is an important local factor in the ability of chorionic gonadotropin (CG) to enhance luteal structure and function in early pregnancy, rhesus monkeys received hCG in a dose-escalating regimen (15-2880 IU twice daily) beginning on Day 9 of the luteal phase of the natural menstrual cycle to simulate the rapid rise in serum CG levels associated with early pregnancy. Some animals were concomitantly treated with the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) inhibitor trilostane (500 mg twice daily) to suppress progesterone production during gonadotropin stimulation. Corpora lutea were removed after 1, 3, 6, and 9 days of treatment (n = 3-4 per group); time-matched control tissues were obtained from untreated animals (n = 3 per group). Treatment with hCG prevented both the decrease in luteal wet weight (p < 0.05) and the histologic indices of luteal regression seen in controls during the menstrual cycle. However, coadministration of the progesterone synthesis inhibitor led to early declines in luteal wet weight (p < 0.05) and luteal cell size compared to treatment with hCG alone. Luteal progesterone receptor (PR) mRNA content increased (p < 0.05), but the percentage of cells staining positive for immunoreactive PR declined (p < 0.05) over the treatment interval in all groups. CG administration alone and in combination with trilostane increased PR staining intensity in some luteal cells within 1 day of treatment; intensely staining cells persisted around vascular elements after 9 days of treatment with hCG+trilostane but not with hCG alone. These data suggest that some, but not all, actions of CG to maintain the primate corpus luteum in early pregnancy are mediated by progesterone via a receptor-mediated pathway.

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

Follicle-stimulating hormone and luteinizing hormone/chorionic gonadotropin stimulation of vascular endothelial growth factor production by macaque granulosa cells from pre- and periovulatory follicles.

Granulosa cells in the ovulatory follicle express messenger ribonucleic acid encoding vascular endothelial growth factor (VEGF), an agent that may mediate the neovascularization of the developing corpus luteum, but it is not known whether luteinizing granulosa cells synthesize and secrete VEGF during the periovulatory interval. Studies were designed to evaluate the effects of an in vivo gonadotropin surge on VEGF production by macaque granulosa cells (study 1) and to test the hypothesis that gonadotropins act directly on granulosa cells to regulate VEGF production (study 2). Monkeys received a regimen of exogenous gonadotropins to promote the development of multiple preovulatory follicles. Nonluteinized granulosa cells (i.e. preovulatory; NLGC) and luteinized granulosa cells (i.e. periovulatory; LGC) were aspirated from follicles before and 27 h after an ovulatory gonadotropin bolus, respectively. Cells were either incubated for 24 h in medium with or without 100 ng/mL hCG (study 1) or cultured for 6 days in medium with or without 100 ng/mL hCG or 0.1, 1, 10, and 100 ng/mL of recombinant human LH (r-hLH) or r-hFSH (study 2). Culture medium was assayed for VEGF and progesterone. In study 1, LGC produced 8-fold greater levels of VEGF than NLGC (899 +/- 471 vs. 111 +/- 26 pg/mL, mean +/- SEM; P < 0.05). In vitro treatment with hCG increased (P < 0.05) VEGF production by NLGC to levels that were not different from the LGC incubated under control conditions. In vivo bolus doses of r-hCG (100 and 1000 IU) and r-hFSH (2500 IU) were equally effective in elevating granulosa cell VEGF production. In study 2, in vitro treatment with r-hFSH, r-hLH, and hCG markedly increased (P < 0.05) VEGF and progesterone production by the NLGC in a dose- and time-dependent manner. By comparison, the three gonadotropins (100 ng/mL dose) only modestly increased VEGF and progesterone production by LGC. These experiments demonstrate a novel role for the midcycle surge of gonadotropin (LH/CG or FSH) in primates to promote VEGF production by granulosa cells in the periovulatory follicle. Further, the data demonstrate that FSH-like as well as LH-like gonadotropins directly stimulate VEGF synthesis by granulosa cells.

Animals↗