Sulfated Glycoprotein-2 mRNA Research Articles

Expression Pattern of Sulfated Glycoprotein-2 (SGP-2) mRNA in Rat Testes Exposed to Endocrine Disruptors

Sulfated glycoprotein-2 (SGP-2) is secreted in Sertoli cells and epididymal epithelial cells and plays important roles in the regulation of spermatogenesis and sperm maturation. To investigate whether endocrine disruptors affect spermatogenesis through an SGP-2-dependent mechanism, daily oral doses of testosterone (50, 200 and 1,000 microg/kg), flutamide (1, 5 and 25 mg/kg), ketoconazole (0.2, 1, 5 and 25 mg/kg), diethylhexylphthalate (10, 50 and 250 mg/kg), nonylphenol (10, 50, 100 and 250 mg/kg), octylphenol (10, 50 and 250 mg/kg), diethylstilbesterol (10, 20 and 40 microg/kg) or corn oil (control) were administered to 5 week-old, male Sprague-Dawley rats for 3 weeks. Following treatment with these endocrine disruptors, testicular expression of SGP-2 mRNA was analyzed using reverse transcription-polymerase chain reaction. Compared with the control, the lowest dose of testosterone (50 microg/kg/day) significantly increased expression of SGP-2 mRNA, whereas 200 and 1,000 microg/kg/day testosterone significantly decreased the expression (P<0.05). Flutamide, ketoconazole, diethylhexylphthalate, nonylphenol, octylphenol and diethylstilbesterol significantly decreased SGP-2 mRNA expression in testes at all doses studied, with the exception of 1 mg/kg/day flutamide (P<0.05). These results suggest that endocrine disruptors might decrease spermatogenesis in testes by decreasing expression of SGP-2 mRNA.

Testicular endocrine function in GH receptor gene disrupted mice.

The consequences of disruption of GH receptor gene in GH receptor knockout mice on testicular function were evaluated. Adult male GH receptor knockout mice and their normal siblings were divided in to two subgroups and treated with either saline or ovine LH (0.3 microg/g BW) in saline. One hour after saline or LH administration, blood was obtained via heart puncture. Plasma IGF-I, LH, FSH, PRL, androstenedione, and testosterone levels were measured by RIAs. Testicular LH and PRL receptor numbers as well as pituitary LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were measured. Also, testicular morphometric analysis was performed. Unlike in normal, wild-type mice, the circulating IGF-I was undetectable in GH receptor knockout mice. The plasma PRL levels were (P<0.01) higher in GH receptor knockout mice than in their normal siblings. The basal LH secretion was similar in normal and GH receptor knockout mice. However, the circulating FSH levels were lower (P<0.001) in GH receptor gene disrupted mice. Administration of LH resulted in a significant (P<0.001) increase in plasma testosterone levels in both GH receptor knockout and normal mice. However, this testosterone response was attenuated (P < 0.01) in GH receptor knockout mice. Plasma androstenedione responses were similar in both GH receptor knockout and normal mice. Testicular LH and PRL receptor numbers were significantly decreased in GH receptor knockout mice. The results of the morphometric analysis of the testis revealed that the Leydig cell volume per testis was reduced in mice with GH receptor gene disruption. The steady-state of LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were not different in GH receptor knockout mice relative to their normal siblings. The present in vivo study demonstrates that in GH receptor knockout mice, LH action on the testis in terms of testosterone secretion is significantly attenuated and suggests that this is due to a decrease in the number of testicular LH receptors. The reduced number of PRL receptors may contribute to the diminished responsiveness of testicular steroidogenesis to LH by decreased ability to convert androstenedione to testosterone. These changes are most likely due to the absence of circulating IGF-I. These findings provide evidence that systemic IGF-I plays a major modulatory role in testicular endocrine function.

Apoptosis in the Rat Penis After Penile Denervation

Purpose Despite the advances in nerve sparing prostatectomy for prostate cancer, some patients develop impotence or subjectively complain of a decrease in penile size. We hypothesized that these clinical observations may be explained by injury to the cavernous nerves resulting in programmed cell death (apoptosis) within the penis. We utilized a rat model of penile denervation in order to demonstrate apoptosis after denervation. Methods and Materials Fifteen male Sprague Dawley rats underwent abdominal exploration and bilateral cavernous neurotomy. Fifteen sham operations were performed as normal controls. The rats were sacrificed on postoperative day 1, 2, 3, 6, and 10 and their penises were harvested. Messenger RNA was extracted and probed on a northern blot for sulfated glycoprotein-2 (SGP-2). SGP-2 is a gene product reported to be elevated in apoptotic tissues. Separate denervated and sham rats were used for DNA extraction (sacrificed postoperative day #2) in order to demonstrate the internucleosomal DNA fragmentation (laddering) found in apoptotic tissues. In addition, in situ histology was performed with ISEL techniques (in situ end labeling) to stain for apoptotic nuclei in denervated rats. Results Northern blot analysis showed a large increase in SGP-2 mRNA expression in the denervated rats with little detected in the sham operated group. DNA extraction studies revealed the presence of internucleosomal DNA fragmentation on agarose gel (a marker for apoptosis) in the denervated group versus intact high molecular weight DNA in the sham rats. In addition, in situ staining of denervated penile erectile tissue demonstrated apoptotic nuclei in the cavernous tissue. Conclusion Apoptosis of penile erectile tissue occurs after denervation of the rat penis. This has not been previously described in the literature and may offer some explanation at the molecular level concerning the mechanism of impotence and/or decrease in penile size after radical prostatectomy.

Epidermal growth factor suppresses renal tubular apoptosis following ureteral obstruction

Objectives. Acute unilateral ureteral obstruction (UUO) results in ipsilateral hydronephrosis characterized by a decrease in epidermal growth factor (EGF) mRNA expression and EGF protein levels in the distal renal tubules. UUO results in programmed cell death with increases in the characteristic markers of apoptosis. To suppress the apoptotic response during UUO, recombinant EGF was administered during renal obstruction and the ensuing molecular and histologic changes were studied. Methods. Mature Sprague-Dawley rats underwent left ureteral obstruction and the kidneys were harvested at 24, 48, and 72 hours. Markers of apoptosis included DNA laddering pattern on agarose gel electrophoresis, in situ gap labeling of fragmented DNA for quantitative apoptotic body determination, polyadenylated mRNA expression of SGP-2, and in situ hybridization for sulfated glycoprotein-2 (SGP-2) mRNA. Studies were repeated in rats following administration of 10, 20, and 40 μg of subcutaneous recombinant EGF on a daily basis after UUO. Results. Subcutaneous injection of EGF into unilaterally obstructed rats promotes renal tubular epithelial cell regeneration, as demonstrated by increased cortical mitotic activity. Systemic EGF supplementation in these unilaterally obstructed rats also resulted in a decrease in the intensity of the DNA laddering pattern associated with renal tubular apoptosis. An in situ labeling procedure to identify apoptotic nuclei in the ureterally obstructed kidneys revealed a 50% reduction in apoptosis after EGF administration. Northern blot analysis and in situ hybridization for SGP-2 mRNA or clusterin gene product also revealed a decreased expression in the obstructed and EGF-treated renal parenchyma. Conclusions. These data suggest that EGF, apart from its known role as a mitogenic substance for renal tubular epithelial cells, is also a critical in vivo renal cell survival factor for the developmentally mature kidney.

Follicular atresia as an apoptotic process: Atresia-associated increase in the ovarian expression of the putative apoptotic marker sulfated glycoprotein-2

To evaluate the possibility that morphologically confirmed/hypophysectomy-induced ovarian follicular atresia, a putative apoptotic process, is coupled to alterations in the steady-state levels of ovarian sulfated glycoprotein-2 (SGP-2) transcripts. Hypophysectomy-induced follicular atresia in immature rats, morphologically confirmed at the light and electron microscopic levels, was correlated with alterations in the steady-state levels of ovarian SGP-2 transcripts as assessed by a solution hybridization/RNase protection assay. Cellular localization was accomplished by in situ hybridization technology. Hypophysectomy of the 24-day-old immature rat, an established precipitant of follicular atresia, led (3 days later) to a significant (P < .05) increase (up to 3.3-fold) in the relative abundance of densitometrically quantified ovarian SGP-2 transcripts compared with age-matched intact controls. Detailed time-course analysis after hypophysectomy revealed significantly (P < .05) increased ovarian SGP-2 mRNA expression as early as 2 days after hypophysectomy; no further increments were noted on days 4 or 8. Light microscopic analysis of comparable ovarian material 4 days after hypophysectomy revealed increased numbers of atretic follicles displaying large numbers of degenerating granulosa cells. Electron microscopic analysis of the degenerating cells of atretic follicles (from hypophysectomized rats) disclosed nuclear condensation and cytoplasmic shrinkage as well as apoptotic bodies at all levels of the granulosa cell layer. In situ hybridization established the granulosa cell of the intact untreated rat as the somatic cell concerned with SGP-2 gene expression. In turn, hypophysectomy led to an increase in SGP-2 expression at the level of the theca-interstitial cell, an effect prevented by the concurrent provision of pregnant mare serum gonadotropin (PMSG). The hypophysectomy-induced increase in ovarian SGP-2 transcripts was similarly reversed (54% inhibition by day 27) by the concomitant provision of FSH, an established antiatretic principle. The delayed administration (day 26) of a single dose of PMSG to rats hypophysectomized on day 24 eliminated the hypophysectomy-induced increase in ovarian SGP-2 transcripts as assessed on day 28. Qualitatively similar but quantitatively more pronounced increments in ovarian SGP-2 gene expression were obtained when atresia was induced by hypophysectomy of PMSG-primed immature rats. These observations establish the immature rat ovary as a site of SGP-2 gene expression and reveal hypophysectomy-induced follicular atresia to result in the up-regulation of ovarian (specifically, theca-interstitial) SGP-2 gene expression, an effect prevented by the concurrent provision of FSH or PMSG. To the extent that SGP-2 is an acceptable apoptotic marker, the present findings support the hypothesis that ovarian follicular atresia may be an apoptotic process.

Primary and secondary genetic responses after folic acid-induced acute renal injury in the mouse.

Folic acid-induced acute renal injury results in dramatic changes in gene expression. Among the genes affected by folic acid treatment are the primary response genes, c-fos and c-myc, which are thought to function to initiate cell cycle events. In this report, changes in the expression of three other genes in response to folic acid injury have been investigated: ornithine decarboxylase, epidermal growth factor (EGF), and sulfated glycoprotein-2 (SGP-2). Renal injury was found to cause a rapid decrease in EGF mRNA, which remained absent for several days after the initial injury, gradually returning to normal levels over an approximately 3-wk regeneration and recovery period. Ornithine decarboxylase mRNA showed a similar decrease. In contrast, folic acid caused a rapid increase in SGP-2 mRNA, which peaked several days after treatment, decreasing to normal levels over the 3-wk period. The mRNAs for the primary response genes were superinduced in the injured kidneys in the presence of the protein synthesis inhibitor cycloheximide. In contrast, the changes in EGF and SGP-2 mRNA levels were blocked by cycloheximide, indicating that these responses required new protein synthesis during the first few hours after folic acid injury. The opposite but parallel responses in the expression of the EGF and SGP-2 genes suggest that their regulation is coupled to the initial injury-induced dedifferentiation and subsequent return to the fully differentiated state.

Apoptosis in decidual tissue regression and reorganization.

During blastocyst implantation, cellular degeneration of decidual tissue spreads from the antimesometrial region to the mesometrial region to accommodate the developing conceptus. To identify the mechanism of decidual regression and its tissue localization, decidual formation was induced in either intact pseudopregnant rats or ovariectomized rats treated with steroids. Antimesometrial and mesometrial cells were separated by elutriation and cultured for 48 h before DNA analysis. Nucleosomal DNA fragmentation was observed in antimesometrial cells, whereas DNA fragmentation was less marked in mesometrial cells. To determine whether a similar pattern of apoptosis occurred during decidual development in vivo, decidua tissues from antimesometrial and mesometrial regions were obtained on days 8-14. Nucleosomal DNA fragmentation was first detected on day 10 in the antimesometrial region, with fragmentation in the mesometrial region delayed by at least 24 h. DNA fragmentation increased in both tissues with time, but was always more pronounced in antimesometrial cells. Sulfated glycoprotein-2 (SGP-2) has been associated with apoptosis, and its expression was examined by Northern analysis. Levels of SGP-2 messenger RNA (mRNA) were detected in the antimesometrial region on day 9 and increased markedly by day 10; mesometrial expression was delayed 24 h. Levels of SGP-2 mRNA in both regions decreased before the onset of DNA fragmentation. In contrast, cathepsin-D expression, detected by immunohistochemistry, was localized to few mesometrial and antimesometrial cells between days 9-12, with all cells showing extensive staining by day 14. To determine whether changes in progesterone control mechanisms initiated decidual apoptosis, plasma progesterone levels were measured by RIA, and progesterone receptor mRNA levels in decidual tissues were examined by reverse transcription-polymerase chain reaction. Both parameters remained unchanged during decidual growth and regression. These results provide biochemical evidence that decidual regression occurs by apoptosis initiated in the antimesometrial region and with progression to the mesometrial region. Apoptotic cell death occurs despite high levels of plasma progesterone and high levels of progesterone receptor message in decidual tissue.

Apoptosis in decidual tissue regression and reorganization

During blastocyst implantation, cellular degeneration of decidual tissue spreads from the antimesometrial region to the mesometrial region to accommodate the developing conceptus. To identify the mechanism of decidual regression and its tissue localization, decidual formation was induced in either intact pseudopregnant rats or ovariectomized rats treated with steroids. Antimesometrial and mesometrial cells were separated by elutriation and cultured for 48 h before DNA analysis. Nucleosomal DNA fragmentation was observed in antimesometrial cells, whereas DNA fragmentation was less marked in mesometrial cells. To determine whether a similar pattern of apoptosis occurred during decidual development in vivo, decidua tissues from antimesometrial and mesometrial regions were obtained on days 8-14. Nucleosomal DNA fragmentation was first detected on day 10 in the antimesometrial region, with fragmentation in the mesometrial region delayed by at least 24 h. DNA fragmentation increased in both tissues with time, but was always more pronounced in antimesometrial cells. Sulfated glycoprotein-2 (SGP-2) has been associated with apoptosis, and its expression was examined by Northern analysis. Levels of SGP-2 messenger RNA (mRNA) were detected in the antimesometrial region on day 9 and increased markedly by day 10; mesometrial expression was delayed 24 h. Levels of SGP-2 mRNA in both regions decreased before the onset of DNA fragmentation. In contrast, cathepsin-D expression, detected by immunohistochemistry, was localized to few mesometrial and antimesometrial cells between days 9-12, with all cells showing extensive staining by day 14. To determine whether changes in progesterone control mechanisms initiated decidual apoptosis, plasma progesterone levels were measured by RIA, and progesterone receptor mRNA levels in decidual tissues were examined by reverse transcription-polymerase chain reaction. Both parameters remained unchanged during decidual growth and regression. These results provide biochemical evidence that decidual regression occurs by apoptosis initiated in the antimesometrial region and with progression to the mesometrial region. Apoptotic cell death occurs despite high levels of plasma progesterone and high levels of progesterone receptor message in decidual tissue.

Renal Tubular Apoptosis after Partial Ureteral Obstruction

Partial ureteral obstruction in the weanling rat leads to hydronephrosis of the ipsilateral kidney and renal cell deletion through the process of programmed cell death known as apoptosis. The apoptotic response following partial ureteral obstruction in weanling Sprague-Dawley rats was studied using the traditional markers of apoptosis, including deoxyribonucleic acid (DNA) laddering pattern on agarose gel electrophoresis, in situ gap labeling of fragmented DNA for quantitative apoptotic body determination, polyadenylated messenger ribonucleic acid (mRNA) expression of sulfated glycoprotein-2, and polyadenylated mRNA expression of epidermal growth factor and transforming growth factor-β. Partial ureteral obstruction resulted in a progressive increase in the intensity of DNA fragmentation associated with apoptosis during the initial 3 weeks. Quantitative apoptotic body counting revealed a 3-fold increase by week 3 of partial obstruction. This increase represented a level of apoptosis, which is 65% of that observed in complete ureteral obstruction. By week 2 of partial obstruction there was a 13-fold increase in the expression of sulfated glycoprotein-2 mRNA, as well as changes in the growth factor environment characterized by a decline in the constitutive expression of epidermal growth factor mRNA and an increase in the expression of transforming growth factor-β mRNA. These altered levels represent changes in expression comparable to those observed during the apoptotic response following complete ureteral obstruction, although the time course is delayed by 2 to 3 weeks.

Increased Expression of Sulfated Glycoprotein-2 and DNA Fragmentation in the Pancreas of Copper-Deficient Rats

Recent morphological studies from our laboratory have shown that copper deficiency-induced pancreatic involution in rats is secondary to apoptosis (M. S. Rao, A. V. Yeldandi, V. Subbarao, and J. K. Reddy, 1993, Am. J. Pathol. 142, 1952-1957). To corroborate the morphological findings, we have examined pancreases from copper-deficient rats for expression of sulfated glycoprotein-2 (SGP-2) mRNA and DNA fragmentation, which are considered as specific markers of apoptosis. Agarose gel electrophoresis of the DNA extracted from pancreas of rats maintained on copper deficient diet (CUDD) for 5 and 7 weeks showed characteristic "ladder" pattern, whereas DNA from control rats and rats maintained on CUDD for 3 weeks showed no fragmentation. These findings correlated well with the histological changes. Northern blot analysis of total RNA revealed a marked increase in the expression of SGP-2 mRNA at 5 weeks followed by a gradual decrease at 6 and 7 weeks. These results further support that the mechanism of copper deficiency-induced pancreatic involution is through apoptosis.

Sulfated glycoprotein-2 mRNA in the rat brain following transient forebrain ischemia

Expression of sulfated glycoprotein-2 (SGP-2) mRNA was studied by in situ hybridization in rat brains submitted to transient forebrain ischemia of 30 min. Induction of this multifunctional protein has been previously observed following diverse types of brain lesions, and an involvement in programmed cell death and synaptic remodelling has been proposed. Ischemia was produced by four-vessel occlusion and followed by various recirculation times ranging from 15 min to 7 days. Up to 6 h after ischemia SGP-2 mRNA did not change in any brain region. After 12 h recirculation, SGP-2 mRNA induction was observed in the stratum lacunosum moleculare of CA1 sector of hippocampus. This induction peaked at 3 days recirculation and then declined. From 24 h recirculation onward, induction also occurred in patchy areas of the cortex, and after 7 days recirculation in the ventral thalamus and in a corona around lesioned parts of the striatum. No induction occurred at any recirculation time in pyramidal neurons of hippocampus or other neuronal populations that are damaged by ischemia. The combination of in situ hybridization with GFAP immunohistochemistry revealed that SGP-2 mRNA was mainly induced in reactive actrocytes. This excludes a direct involvement in ischemic neuronal death and supports the possible participation in the post-lesional reorganization of the tissue.

A new target for growth hormone releasing-hormone action in rat: the Sertoli cell.

A GHRH-like mRNA and peptide (t-GHRH) have been detected in rat and human testis. In rat, t-GHRH mRNA is localized to developing spermatogenic cells. We predicted that the most likely target cell of t-GHRH action would be the Sertoli cell. To test this prediction, we evaluated GHRH action on Sertoli cell function. Rat GHRH at a concentration of 10 nM or 100 nM stimulated cAMP production 2-fold over control levels after a 30 min incubation. This stimulation was obliterated by preincubation with a 10-fold excess of the GHRH antagonist (N-Ac-Tyr1, D-Arg2)-GRF(1-29)-NH2. The effect of treatment with [His1,Nle27]GHRH(1-32)-NH2, a GHRH analog, on Sertoli cell mRNAs was also assessed. Treatment with the analog significantly increased levels of c-fos and steel factor (the product of the Steel gene, also termed SCF) mRNAs above controls, but had no effect on sulfated glycoprotein-2 mRNA. We conclude that GHRH acts via adenylate cyclase to modulate specific Sertoli cell products, possibly as part of a network of local interacting factors controlling Sertoli and germ cell function.

A new target for growth hormone releasing-hormone action in rat: the Sertoli cell

A GHRH-like mRNA and peptide (t-GHRH) have been detected in rat and human testis. In rat, t-GHRH mRNA is localized to developing spermatogenic cells. We predicted that the most likely target cell of t-GHRH action would be the Sertoli cell. To test this prediction, we evaluated GHRH action on Sertoli cell function. Rat GHRH at a concentration of 10 nM or 100 nM stimulated cAMP production 2-fold over control levels after a 30 min incubation. This stimulation was obliterated by preincubation with a 10-fold excess of the GHRH antagonist (N-Ac-Tyr1, D-Arg2)-GRF(1-29)-NH2. The effect of treatment with [His1,Nle27]GHRH(1-32)-NH2, a GHRH analog, on Sertoli cell mRNAs was also assessed. Treatment with the analog significantly increased levels of c-fos and steel factor (the product of the Steel gene, also termed SCF) mRNAs above controls, but had no effect on sulfated glycoprotein-2 mRNA. We conclude that GHRH acts via adenylate cyclase to modulate specific Sertoli cell products, possibly as part of a network of local interacting factors controlling Sertoli and germ cell function.

Localization of sulfated glycoprotein-2/clusterin mRNA in the rat brain by in situ hybridization.

Sulfated glycoprotein-2 (SGP-2) gene expression seems to be constitutively expressed in a variety of tissues and organs, although levels of expression vary widely from one tissue to the other. SGP-2, also known as clusterin, has been reported to be expressed in the central nervous system (CNS). Some possible roles for brain SGP-2 have been postulated. In order to provide a substrate for a better understanding of the functions of this glycoprotein in the CNS, we investigated the detailed anatomical and cellular distribution of SGP-2 mRNA in the adult rat brain as well as the variation in its cellular expression after excitotoxin lesion. Transcripts for SGP-2 were found to be distributed throughout the rat CNS, although regional differences in their prevalence were readily observed. The ependymal lining of the ventricles showed the highest level of expression followed by various gray matter areas, some of which contained very intensively labeled cells. These cells were mostly found among several hypothalamic and brainstem nuclei, the habenular complex, as well as in the ventral horn of the spinal cord, which displayed striking hybridization signals over motoneurons. Occasional cells expressing high levels of SGP-2 transcripts were found in fiber tracts. Highly SGP-2 mRNA-positive resting glial cells were mainly located near the glial limitans and blood vessels. Two areas of relatively low constitutive SGP-2 mRNA expression are shown to produce strong hybridization signals 10 days after the local administration of the excitotoxin kainic acid. This overexpression of SGP-2 transcripts appears to involve GFAP-positive cells. Taken together, these results indicate that in the intact adult rat CNS, various cell populations, including neurons, constitutively express SGP-2 transcripts, whereas in the injured brain, reactive astrocytes become the major producers.

Seizure activity causes a rapid increase in sulfated glycoprotein-2 messenger RNA in the adult but not the neonatal rat brain

The present study investigated the changes in sulfated glycoprotein-2 (SGP-2) messenger RNA at various times following kainic acid-induced seizure onset in adult and neonatal rat brain. Double labelling using immunostaining of the astrocyte-specific glial fibrillary acidic protein indicated that SGP-2 expression was rapidly and transiently increased in granule cells of the dentate gyrus up to 8 hours after seizure onset. Thereafter, and up to 7 days following seizure onset, the majority of cells exhibiting increased SGP-2 expression were astrocytes located in the molecular layer of the dentate gyrus and in the alveus, as well as in regions adjacent to CA3 and CA1 pyramidal cells. No increase in SGP-2 mRNA was detected in pyramidal neurons selectively damaged by KA. In addition, increased expression of SGP-2 following KA administration was not observed in neonatal rat hippocampus prior to postnatal day 21. The results argue against a role for SGP-2 in KA-induced neuronal death and demonstrate a surprisingly rapid increase in astroglial gene expression following seizure activity, thus supporting a role for SGP-2 in synaptic plasticity.

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

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

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

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

Regulation of sulfated glycoprotein-2 (clusterin) messenger ribonucleic acid in the rat epididymis.

Sulfated glycoprotein-2 (SGP-2) is secreted by the principal cells of the caput epididymidis and binds to spermatozoa as they transit through this segment. The regulation of SGP-2 in the epididymis is poorly understood. The objectives of these studies were to determine if SGP-2 messenger RNA (mRNA) concentrations in the epididymis are regulated by testosterone or during postnatal development. Northern blot analysis was done using denatured plasmid containing a complementary DNA insert for rat SGP-2. A single 2.1-kilobase transcript was present throughout the epididymis. SGP-2 mRNA concentrations were highest in the caput followed by the initial segment, the cauda, and the corpus epididymidis. To determine if androgens regulate SGP-2 mRNA concentrations, adult rats were bilaterally orchidectomized and testosterone was replaced for 7 days using steroid-filled capsules measuring 2.5 or 18.6 cm. In the initial segment and the caput epididymidis, neither orchidectomy nor testosterone replacement, at either dose, had any effect on SGP-2 mRNA concentrations. In the corpus and cauda epididymidis, bilateral orchidectomy resulted in a 3.5- and 9.4-fold increase, respectively, in SGP-2 mRNA concentrations, whereas testosterone replacement caused a dose-dependent decrease in SGP-2 mRNA concentrations. Unilateral orchidectomy was done to determine if SGP-2 mRNA concentrations are dependent on testicular factors released in the lumen of the epididymis. In the corpus and the cauda epididymidis, unilateral orchidectomy resulted in elevated SGP-2 mRNA concentrations in the ipsilateral epididymis. There were no changes in SGP-2 mRNA concentrations in the initial segment and caput epididymidis. These results provide complementary evidence that the message for SGP-2 is differentially regulated along the epididymis. During postnatal development SGP-2 mRNA concentrations in the caput-corpus epididymidis increased dramatically between 14 and 21 days as well as between 49 and 63 days. Interestingly, between 28 and 42 days, when serum testosterone concentrations are increasing, there was no change in the concentration of SGP-2 mRNA in the caput-corpus epididymidis. Similar results were observed in the cauda epididymidis with the exception that between 28 and 42 days, there was a dramatic decrease in SGP-2 mRNA in the cauda epididymidis. Together these experiments demonstrate that the regulation of SGP-2 mRNA concentrations is segment specific. In the initial segment and caput epididymidis there is no apparent regulation of SGP-2 by testicular factors,whereas in the corpus and cauda epididymidis testosterone can repress SGP-2 mRNA concentrations.

Ovarian 3 beta-hydroxysteroid dehydrogenase and sulfated glycoprotein-2 gene expression are differentially regulated by the induction of ovulation, pseudopregnancy, and luteolysis in the immature rat.

The present studies were conducted to elucidate the effects of gonadotropin-induced alterations in ovarian status on expression of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD), an enzyme which plays a crucial role in steroidogenesis, and sulfated glycoprotein-2 (SGP-2), a heterodimeric protein which is highly expressed by cells undergoing programmed death (i.e. apoptosis). Prepubescent female rats were used to reduce the influence of endogenous gonadotropins and to avoid the presence of preexisting, degenerating corpora lutea in the ovaries. 3 beta-HSD, cholesterol side-chain cleavage cytochrome P450, and SGP-2 messenger RNA (mRNA) levels were measured by Northern analysis of total ovarian RNA. Rats which received PMSG (20 IU) followed 48 h later by human CG (hCG) (10 IU) to induce ovulation and pseudopregnancy exhibited a significant increase in ovarian 3 beta-HSD mRNA 1 day later (164%, P less than 0.01 vs. saline control). The most dramatic change in 3 beta-HSD expression was the rise seen 2 days after hCG (262%, P less than 0.01), after which levels remain constantly elevated throughout pseudopregnancy. In contrast, ovarian cholesterol side-chain cleavage cytochrome P450 mRNA was greatly elevated (i.e. 15-fold) 48 h after PMSG treatment alone (P less than 0.01). Thus, gonadotropic stimulation which induces ovulation and luteogenesis is required to observe a potent stimulatory effect on ovarian 3 beta-HSD expression. The slow time course of induction is indicative of a differentiation-dependent expression. These observations are consistent with luteal cell expression of the 3 beta-HSD gene and suggest that this expression is correlated with the high progestin secretion and 3 beta-HSD activity characteristic of luteal cells. Interestingly, the pattern of regulation of ovarian SGP-2 expression was markedly different than that observed for 3 beta-HSD. PMSG treatment alone (48 h), and in combination with hCG, dramatically reduced SGP-2 mRNA to 12-27% of controls (P less than 0.01). SGP-2 levels were not elevated until 7 days after hCG; levels then remained constant through day 14 of pseudopregnancy. Since luteal progesterone secretion begins to diminish 5-7 days after hCG, the increased expression of SGP-2 on day 7 may be related to the initiation of the regression/degeneration of luteal cells which occurs during luteolysis. Thus, this study demonstrates that alterations in SGP-2 expression by the ovary may precede or occur simultaneously with cellular events initiating luteolysis and suggests a role for this glycoprotein as an early marker for luteolysis and implicates it in yet another instance of programmed cell death.

Developmental Regulation of Ornithine Decarboxylase (ODC) in Rat Testis: Comparison of Changes in ODC Activity with Changes in ODC mRNA Levels during Testicular Maturation1

These studies were undertaken to analyze the changes in testicular ornithine decarboxylase (ODC) mRNA levels and ODC activity in rats from birth to maturity. Levels of ODC mRNA were initially low in animals aged 10-17 days. Beginning at 21 days, ODC mRNA levels began to rise, reaching maximal levels by 40 days (p less than 0.01). The size of the 2.2- and 2.6-kb ODC mRNAs did not appear to change with age, as determined by Northern blot analysis. The increase in ODC mRNA that began at 21 days paralleled the increase in testis weight. This increase in ODC mRNA preceded the appearance of rat protamine-1 mRNA, a germ cell-specific mRNA found in round spermatids, which was first detected on Day 40. In contrast, levels of sulfated glycoprotein-2 mRNA, which, in the testis, is found exclusively in Sertoli cells, were highest at Day 17 and thereafter declined gradually with age. Unlike the increase in ODC mRNA levels, ODC activity was highest in 10-day-old animals and thereafter declined steadily with age, reaching minimal levels by 40 days (p less than 0.01). Thus, the increase in testicular ODC mRNA levels was in marked contrast to the decrease in testicular ODC activity. Incubation of cytosolic extract from 40-day-old animals with that from 10- or 17-day-old animals inhibited ODC activity approximately 50%, when compared to cytosols from 10- or 17-day-old animals. Dialysis of cytosol from 40-day-old animals prior to incubation with cytosol from 10-day-old animals relieved this inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)