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.

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.

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

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.

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.

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

Summary. Theca cells were collected from the second largest preovulatory follicle. Chelation of extracellular calcium with EGTA attenuated LH (10 ng)-induced androstenedione production by theca cells, and this effect was more pronounced in calcium-deficient than in calcium-replete incubation medium. Incubation of theca cells with steroidogenic agonists in the presence of the calcium channel blocker verapamil (100μm) suppressed androstenedione production stimulated by LH (a 57% decrease), the adenylate cyclase activator forskolin (a 59% decrease) and the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP (a 61% decrease). Furthermore, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a putative inhibitor of intracellular calcium mobilization, suppressed LH-induced androstenedione production in a dose-dependent fashion.

The calmodulin inhibitors trifluoperazine (100μm) and R24571 (50μm) inhibited androstenedione production stimulated by hormonal (LH) and non-hormonal (forskolin, 8-bromo-cAMP) agonists (decreases ranging from 76 to 98%). While increasing the intracellular calcium ion concentrations with the calcium ionophore A23187 did not affect basal concentrations of androstenedione, treatment of LH-stimulated cells with the ionophore caused dose-dependent inhibition of androstenedione production; these effects were enhanced by coincubation with phorbol 12-myristate 13-acetate (a known activator of protein kinase C). We conclude that the mobilization of calcium is critical for agonist-stimulated steroidogenesis in hen theca cells, apparently requiring the interaction of calcium with its binding protein, calmodulin. Furthermore, increased cytosolic calcium concentrations may be involved in the suppression of androstenedione production, possibly as a result of an interaction with protein kinase C.

Keywords: androstenedione; calcium; calmodulin; hen; ovary; theca

Animal Physiology and Genetics Institute, and Agricultural Environmental Quality Institute, U.S. Department of Agriculture, Beltsville, Maryland 20705, U.S.A.

(Received 20th May 1975)

Numerous studies have demonstrated the value of using heterospermic inseminations for comparing the fertilizing capacity of males (Edwards, 1955; Beatty, 1957, 1960; Beatty et al., 1969; Stewart et al., 1974; Overstreet & Adams, 1971; Martin & Reimers, 1973) and for assessing sperm treatments (Roche et al., 1968; Miller et al., 1969; Dziuk, 1970; O'Reilly et al., 1972). Overstreet & Adams (1971) and Bedford & Overstreet (1972) showed that X-irradiation of rabbit spermatozoa in vitro effectively 'marked' the sperm nucleus without affecting the fertilizing capacity. Ova fertilized by the 'marked' spermatozoa have retarded cleavage. The 'marked' spermatozoa were mixed with unmarked spermatozoa from the same ejaculate and used to test the comparative fertilizing capacity after different sperm treatments were superimposed on the two sperm populations.

Taber & Borkovec (1969)

Summary.

Scrotal testes from control and unilaterally cryptorchid rats and rabbits and abdominal testes from animals unilaterally or bilaterally cryptorchid for 6 days were used in this study. Total cholesterol rose (P<0·05) in all abdominal testes as a result of increased (P<0·05) levels of esterified cholesterol. Fatty acids of the scrotal testes from unilaterally cryptorchid animals were only slightly changed by treatment. Changes in fatty acid esters in abdominal testes of unilaterally cryptorchid animals of both species were similar. These changes consisted of increases in most fatty acids with a chain length of 14, 16 and 18, both saturated and unsaturated. Abdominal testes of bilaterally cryptorchid rabbits differed from those of unilateral cryptorchids in that proportions of long chain fatty acids were more consistently increased in the former. Abdominal testes of the bilaterally cryptorchid rats also differed from the unilateral cryptorchids but less than in the rabbit. Differences in hormonal levels in the unilateral and bilateral cryptorchids are suggested as the most probable cause of difference in the abdominal testes of these groups.

A growing body of literature provides evidence of a prominent role for bone morphogenetic proteins (BMPs) in regulating various stages of ovarian follicle development. Several actions for BMP6 have been previously reported in the hen ovary, yet only within postselection (preovulatory) follicles. The initial hypothesis tested herein is that BMP6 increases FSH receptor (FSHR) mRNA expression within the granulosa layer of prehierarchal (6–8 mm) follicles (6–8 GC). BMP6 mRNA is expressed at higher levels within undifferentiated (1–8 mm) follicles compared with selected (≥9 mm) follicles. Recombinant human (rh) BMP6 initiates SMAD1, 5, 8 signaling in cultured 6–8 GC and promotes FSHR mRNA expression in a dose-related fashion. In addition, a 21 h preculture with rhBMP6 followed by a 3 h challenge with FSH increases cAMP accumulation, STAR (StAR) expression, and progesterone production. Interestingly, rhBMP6 also increases expression of anti-Müllerian hormone (AMH) mRNA in cultured 6–8 GC. This related BMP family member has previously been implicated in negatively regulating FSH responsiveness during follicle development. Considering these data, we propose that among the paracrine and/or autocrine actions of BMP6 within prehierarchal follicles is the maintenance of both FSHR and AMH mRNA expression. We predict that before follicle selection, one action of AMH within granulosa cells from 6 to 8 mm follicles is to help suppress FSHR signaling and prevent premature granulosa cell differentiation. At the time of selection, we speculate that the yet undefined signal directly responsible for selection initiates FSH responsiveness. As a result, FSH signaling suppresses AMH expression and initiates the differentiation of granulosa within the selected follicle.