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R. L. Johnson and E. Kapsalis

The reproductive performance of 760 free-ranging female rhesus monkeys (Macaca mulatta), 168 of whom were 20 years of age and older at the time observations were begun, was assessed. The monkeys were resident on Raccoon Key or Key Lois, two islands located in the Florida Keys, USA. During 1992 and 1993, live birth rates generally declined with age among the Raccoon and Lois females aged eight years and older. This age-related deterioration of female fertility was the result of proportionately more younger females bearing live young during successive birth seasons, and proportionately more older females experiencing an inability to bear live offspring even after a barren year. It is suggested that (1) older females may be more strongly inhibited by the suckling stimulus than are their younger peers, and (2) the risk of a permanent loss of fecundity increases with each additional year of life or parturition. The live birth rates of females aged 16–24 years were greater on Raccoon Key than they were on Key Lois, because the Raccoon females within this age range were more successful at bearing live offspring during successive birth seasons; the reason for this difference could not be determined. Inter-population differences in both the body condition of the females and the severity of female–female competition for access to males were not considered to be plausible explanations. It is possible that the difference in female fertility between the islands is the result of the greater age of the adult males on Key Lois, or the phytochemicals eaten by the females on Raccoon Key.

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L. Johnson and H. B. Nguyen

Summary. Stereological methods were employed in two experiments with adult stallions: (1) to confirm seasonal variation in number of Sertoli cells and (2) to characterize the annual cycle of the Sertoli cell population. In the first experiment, testes from 28 adult (4–20 years old) horses obtained in the non-breeding season (December—January) were compared to testes from 28 adult horses in the breeding season (June—July). Sertoli cell numbers were calculated from the nuclear volume density, parenchymal volume, and volume of an individual Sertoli cell nucleus determined by reconstruction of serial sections or from average height and width measurements. The number of Sertoli cells per testis was significantly greater in the breeding season. In a second experiment involving 43–48 adult horses in each 3-month period, the Sertoli cell population was higher (P < 0.05) in May—July than other periods and higher (P < 0.01) than in November—January. These combined studies confirm seasonal differences in the Sertoli cell numbers per testis and define the annual cycle of the Sertoli cell population in adult stallions.

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L. Johnson and D. L. Thompson Jr

Summary. Testes from 47 adult (4–20 years) stallions obtained in November–January (non-breeding season) and 41 adult stallions obtained in May–July (breeding season) were perfused with glutaraldehyde, placed in osmium and embedded in Epon 812. Percentage Leydig cell cytoplasm or nuclei in the testis was determined by point counting of 0·5 μm sections under bright-field microscopy. Testes from 6 randomly selected horses per season were processed for electron microscopy. The volume (ml) of SER/testis was calculated from the % SER in the cytoplasm % Leydig cell cytoplasm, and parenchymal volume. Number of Leydig cells was calculated from the % nuclei, parenchymal volume, histological correction factor, and volume of single nucleus. Intratesticular testosterone content was determined from the contralateral testis by radioimmunoassay. The volume of SER/g and testosterone/g tended to be higher in the breeding than non-breeding season. Leydig cell number/g, volume of SER/testis, testosterone/testis, and Leydig cell number/testis were significantly greater in the breeding than in the non-breeding season. Volume of SER/testis and testosterone/testis were related significantly to the cell number/testis, and SER/testis was related (P < 0·05) to testosterone/testis. These results emphasize the importance of seasonal changes in the number of Leydig cells on the amount of SER available to produce testosterone and on testosterone content/testis in the stallion.

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L. A. KRAFT and A. D. JOHNSON

Summary.

After unilateral separation of the rat epididymis from the testis, the metabolism of various substrates in vitro by tissue from the attached and separated caput and cauda epididymidis at 7 and 28 days after surgery was determined by radiorespirometry. Hourly collections of 14CO2 were made during 5-hr incubations. The patterns of 14CO2 evolution from glucose indicated that most of the metabolic activity followed the Embden-Meyerhof glycolytic and the Krebs cycle respiration pathways. The alteration of the rate of glycolysis was always greater than that of respiration. In all samples, the metabolism of [2-14C]glucose was approximately equal to that of [6-14C]glucose (G-6) and less than that of [1-14C]glucose (G-1). Pentose cycle activity was indicated in all tissues from the caput and cauda epididymidis by the preferential utilization of G-1 over G-6. At 7 and 28 days after surgery, respectively, the G-1 :G-6 ratios of 14CO2 evolution after incubation for 2 hr were 9·75 and 7·79 for the separated caput, 5·17 and 2·66 for the intact caput, 3·11 and 2·52 for the separated cauda and 3·73 and 2·84 for the attached cauda epididymidis. Although epididymal separation did not affect the metabolism of [U-14C]glucose or [U-14C]fructose, glucose appeared to be a more important epididymal substrate than fructose.

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Dori C Woods and A L Johnson

While there is accumulating evidence that mitogen-activated protein kinase/Erk and protein kinase C (PKC) signaling inhibits premature differentiation of granulosa cells in hen prehierarchal follicles, it has only recently been established that these signaling pathways play an important facilitory role in promoting steroidogenesis in differentiated granulosa cells from preovulatory follicles. The present studies were conducted with differentiated granulosa cells to establish the ability of LH to initiate PKC activity, and the subsequent requirement for PKC activity in promoting the ErbB/Erk signaling cascade that ultimately facilitates LH-induced progesterone production. Incubation of differentiated granulosa cells with LH increases PKC activity within 15 min, and latently promotes Erk phosphorylation (P-Erk) by 180 min. Inhibition of PKC activity with GF109203X attenuates LH- and 8-bromo-cAMP (8-br-cAMP)-induced P-Erk, but not P-Erk promoted by an epidermal growth factor (EGF) family ligand (e.g., transforming growth factor α). Importantly, inhibition of PKC activity also blocks the LH-induced increase in the autocrine expression of mRNA encoding the EGF family ligands, such as EGF, amphiregulin, and betacellulin. Furthermore, inhibition of EGF ligand shedding at the level of the cell membrane using the matrix metalloprotease activity inhibitor, GM6001, prevents both LH- and 8-br-cAMP-induced P-Erk and progesterone production. These findings provide evidence for a facilitory role of PKC and ErbB/Erk signaling in LH-induced progesterone production, place the requirement for PKC activation upstream of ErbB/Erk activity, and demonstrate for the first time in a non-mammalian vertebrate the requirement for PKC activity in LH-induced expression of EGF family ligands in granulosa cells.

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Morgan J Haugen and A L Johnson

Prior to follicle selection into the preovulatory hierarchy, hen granulosa cells from prehierarchal follicles remain undifferentiated, as defined in part by the virtual absence of LHR mRNA expression and inability to produce progesterone. It has previously been proposed that prior to follicle selection, granulosa cells are actively maintained in an undifferentiated state by epidermal growth factor receptor ligands (EGFRL) signaling via the MAP kinase/extracellular regulated kinase pathway. Moreover, there is recent evidence that EGFRL/MAP kinase signaling modulates FSH receptor (FSHR) transcription, in part, via inhibitor of differentiation/DNA-binding (ID) proteins. In the present studies with undifferentiated granulosa, recombinant human (rh) bone morphogenetic protein 2 (BMP2) induced the phosphorylation of SMAD1/5/8, and blocked transforming growth factor β and FSH-induced FSHR expression and progesterone production. Significantly, BMP2 rapidly induced mRNAs encoding betacellulin and EGF, plus ID proteins (ID1, ID3, and ID4). Alternatively, the bioactivity of BMPs can be modulated by one or more BMP antagonists, including noggin (NOG). NOG mRNA is expressed by both hen granulosa and theca tissues from prehierarchal follicles. Pretreatment of cultured granulosa with rh NOG reversed both the stimulatory effects of BMP2 on ID1, ID3, and ID4 expression and the inhibitory effects of BMP2 on FSHR mRNA levels and progesterone production. Collectively, these data provide evidence that prior to follicle selection, BMP2 signaling contributes toward maintaining granulosa cells in an undifferentiated state. The actions of BMP2 are, at least in part, mediated indirectly via enhanced EGFRL expression and ERBB receptor-mediated MAP kinase signaling, and can be modulated by the autocrine/paracrine production of NOG.

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F. A. Johnson and N. L. Poyser

Summary. Melittin, an activator of phospholipase (PL) A-2, increased the outputs of prostaglandin (PG) F-2α and 6-keto-PGF-1α, but not of PGE-2, from Day-7 guineapig uterus superfused in vitro. Reducing the extracellular calcium concentration (by omitting calcium chloride from the superfusing fluid) partially inhibited the stimulatory effect of melittin on uterine PG production. TMB-8 (an intracellular calcium antagonist) completely prevented the stimulation of PGF-2α and 6-keto-PGF-1α output by melittin, although the production of both PGs tended to increase after stopping the melittin and TMB-8 treatments. TMB-8 also inhibited the increases in outputs of PGF-2α, 6-keto-PGF-1α and PGE-2 and prevented contraction of the uterus induced by exogenous PLA-2. Trifluoperazine (a calmodulin antagonist) had no inhibitory effect on the increases in outputs of PGF-2α and 6-keto-PGF-1α produced by melittin; it potentiated the stimulatory effect of melittin on 6-keto-PGF-1α output and allowed melittin to increase PGE-2 output. When melittin was applied twice to the superfused uterus with an interval of 1 h between each treatment, partial refractoriness of the responses to melittin was seen: the magnitudes of the increases in PGF-2α and 6-keto-PGF-1α outputs were 40–50% less after the second treatment than after the first treatment. These results show that melittin stimulates the synthesis of PGF-2α and PGI-2 (measured as 6-keto-PGF-1α) in guinea-pig uterus by mechanisms which are calcium dependent. The results are compatible with there being a protein of similar functional activity to melittin in the guinea-pig uterus, which may be involved in the stimulation of endometrial PGF-2α synthesis.

Keywords: melittin; TMB-8; trifluoperazine; prostaglandins; guinea-pig; uterus

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J. M. Levorse and A. L. Johnson

Studies were conducted to evaluate the regulation of steroid production in dispersed cells from ovarian stromal tissue from 5- to 8-week-old-pullets (IM cells) and laying hens (MAT cells). Short-term incubation of IM and MAT cells with ovine (o) LH resulted in a dose-dependent increase in progesterone, androstenedione and oestradiol production; progesterone production was greater in MAT cells than in IM cells (P < 0.05) in response to 2–200 ng oLH ml−1, whereas androstenedione and oestradiol production was greater in MAT cells following treatment with 20 and 200 ng oLH ml−1 (P < 0.05). In both cell populations the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (1 and 10 mmol l−1) stimulated progesterone and androstenedione production, whereas oLH (200 ng ml−1) and forskolin (1–10 μmol l−1) promoted cAMP accumulation (P < 0.05 compared with basal values). However, treatment with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), did not alter basal or oLH-stimulated cAMP accumulation or progesterone production in either IM or MAT cells (P > 0.10). PMA did, however, inhibit agonist-induced androstenedione production (P < 0.05); co-treatment with the calcium ionophore A23187 potentiated this inhibitory effect. Finally, treatment with transforming growth factor-α (TGF-α; 1.8–18 pmol l−1) did not affect basal or oLH-stimulated progesterone or androstenedione production by IM cells, MAT cells, theca cells from 6–8 mm follicles or theca cells from the second largest (F2) follicle (P > 0.10). We conclude that LH-stimulated steroid production is greater in MAT cells than in IM cells; production of steroids at both stages occurs, at least in part, via the adenylyl cyclase/cyclic AMP second messenger pathway. However, we propose that activation of protein kinase C can inhibit agonist-induced cytochrome P450 17α-hydroxylase, but not cytochrome P450 cholesterol side-chain cleavage, activity in stromal cells. Finally, steroidogenesis in stromal tissue from 5- to 8-week-old pullet and laying hen ovaries is regulated by hormonal and cellular mechanisms most comparable to those that modulate steroidogenesis in theca cells from 6- to 8-mm and F2 follicles.

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J. M. Johnson and L. C. Ellis

Summary. PG synthetase activity was assessed histochemically in the reproductive tract of male rats. Moderate activity was observed in tails of spermatozoa within the corpus and cauda epididymidis but there was no activity in the caput epididymidis or the seminiferous tubules. The sperm tail activity was maximal for cells within the vas deferens. PG synthetase activity was also observed in individual adipose cells adhering to the testicular capsule, epididymis and vas deferens, and in isolated interstitial cells of the testis and the caput, corpus and cauda epididymidis. Specific cells in the capsules of the testes, epididymis and vas deferens also produced PGs. The activity observed in the interstitial cells of the testis and the caput epididymidis was less than that for the other tissues in terms of the proportion of possible cells. The demonstration of PG synthetase activity paralleled the known loss of arachidonic acid from the phospholipids of the spermatozoa as they pass through the male tract. Endogenous substrate was not limiting in the assay system, even in the testis and caput epididymidis where PG synthesis was not normally observed, indicating that a PG synthesis inhibitor may be present in these two tissues. PG synthetase activity within teased seminiferous tubules was markedly increased by physical trauma. Indomethacin diminished but did not eliminate synthesis.

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A. L. Johnson, S. E. Becker, and M. L. Roma

Summary. Standard bred mares that were cycling normally were treated beginning on Days 9 or 10 of the oestrous cycle with repeated pulses of GnRH (20 μg/h) and/or a single injection of prostaglandin (PG)F-2α (alfaprostol, 3 mg), and were subsequently bled and palpated daily until the next ovulation. GnRH treatment increased serum concentrations of LH and progesterone at 4 days after the start of treatment compared to controls. The combination of PGF-2α + GnRH treatment resulted in an immediate decline in serum progesterone values, and subsequently decreased the interval to next ovulation by 4·5 days compared to controls. Mean serum concentrations of FSH were not different among treatment groups 4 days after the start of treatment, and there was a consistent trend among all treatment groups for decreasing concentrations of FSH within the 6 days before ovulation. We conclude that, under our experimental conditions, pulsatile administration of GnRH provides a short-term luteotrophic stimulus, probably by the elevation in serum LH, but that this stimulus cannot indefinitely prevent the luteolytic effects of exogenously administered PGF-2α. Although GnRH treatment combined with PGF-2α injection hastened the impending ovulation, this regimen was no more effective than PGF-2α treatment alone.

Keywords: mare; ovulation; LH; FSH; progesterone; corpus luteum; PGF-2α