Kevin J. Barr

Failure of Spermatogenesis in Mice Lacking Connexin43

Abstract Connexin43 (Cx43), a gap junction protein encoded by the Gja1 gene, is expressed in several cell types of the testis. Cx43 gap junctions couple Sertoli cells with each other, Leydig cells with each other, and spermatogonia/spermatocytes with Sertoli cells. To investigate the role of this communication pathway in spermatogenesis, we studied postnatal testis development in mice lacking Cx43. Because such mice die shortly after birth, it was necessary to graft testes from null mutant fetuses under the kidney capsules of adult males for up to 3 wk. Grafted wild-type testes were used as controls. In our initial experiments with wild-type testes, histological examination indicated that the development of grafted testes kept pace with that of nongrafted testes in terms of the onset of meiosis, but this development required the presence of the host gonads. When excised grafts were stimulated in vitro with cAMP or LH, there was no significant difference in androgen production between null mutant and wild-type testes, indicating that the absence of Cx43 had not compromised steroidogenesis. Previous research has shown that Cx43 null mutant neonates have a germ cell deficiency that arises during fetal life, and our analysis of grafted testes demonstrated that this deficiency persists postnatally, giving rise to a “Sertoli cell only” phenotype. These results indicate that intercellular communication via Cx43 channels is required for postnatal expansion of the male germ line.

MULTIPLE MEMBERS OF THE CONNEXIN GENE FAMILY PARTICIPATE IN PREIMPLANTATION DEVELOPMENT OF THE MOUSE

The connexin gene family, of which there are at least 12 members in rodents, encodes the protein subunits intercellular membrane channels (gap junction channels). Because of the diverse structural and biophysical properties exhibited by the different connexins, it has been proposed that each may play a unique role in development or homeostasis. We have begun to test this hypothesis in the preimplantation mouse embryo in which de novo gap junction assembly is a developmentally regulated event. As a first step, we have used reverse transcription-polymerase chain reaction (RT-PCR) to determine the connexin mRNA phenotype of mouse blastocysts, and have identified transcripts of connexins 30.3, 31, 31.1, 40, 43, and 45. Quantitative measurements indicated that all six of these connexin genes are transcribed after fertilization. They can be divided into two groups with respect to the timing of mRNA accumulation: Cx31, Cx43, and Cx45 mRNAs accumulate continuously from the two- or four-cell stage, whereas Cx30.3, Cx31.1, and Cx40 mRNAs accumulate beginning in the eight-cell stage. All six mRNAs were found to co-sediment with polyribosomes from their time of first appearance, indicating that all six are translated. The expression of Cx31.1 and Cx40 was examined by confocal immunofluorescence microscopy; whereas both could be detected in compacting embryos, only Cx31.1 could be seen in punctate membrane foci indicative of gap junctions. Taken together with other results (published or submitted), our findings indicate that at least four connexins (Cx31, 31.1, 43 and 45) contribute to gap junctions in preimplantation development. The expression of multiple connexin genes during this early period of embryogenesis (when there are only two distinct cell types) raises questions about the functional significance of connexin diversity in this context.

Interplay between paracrine signaling and gap junctional communication in ovarian follicles

Intercellular communication is required for ovarian folliculogenesis. This is apparent in mice lacking connexin43 (Cx43, a gap junction protein strongly expressed in granulosa cells), or growth/differentiation factor-9 (GDF9, an oocyte-specific growth factor that stimulates granulosa cell proliferation and differentiation), or in mice expressing a mutant form of Kit ligand (KITL, a paracrine factor that, in the ovary, is secreted by granulosa cells to stimulate oocyte growth). In all of these mutant lines, follicle growth is impaired suggesting a possible interaction between paracrine signaling and gap junctional communication. To assess this possibility, we analyzed gene expression in mutant ovaries. Despite the lack of gap junctional coupling between granulosa cells of Cx43 null mutant ovaries, expression of the genes encoding KITL and its receptor, KIT, is maintained. Furthermore, GDF9 expression is maintained. In GDF9 null mutant ovaries, there is no apparent change in Cx43 expression and, correspondingly, the granulosa cells remain coupled. There is also no increase in granulosa cell apoptosis in ovaries lacking Cx43 or GDF9. Staining for proliferating cell nuclear antigen (PCNA) revealed that the granulosa cells of Cx43 null mutant ovaries have a reduced frequency of DNA synthesis. Using both radiolabeled thymidine incorporation and PCNA staining in vitro, we showed that recombinant GDF9 could restore the proliferation of coupling-deficient granulosa cells to the level of control cells. These results indicate that impaired folliculogenesis in mice lacking Cx43 is due at least in part to reduced responsiveness of granulosa cells to oocyte-derived GDF9, indicating an interaction between these two modes of intercellular communication.

Na,K-ATPase from mice lacking the subunit (FXYD2) exhibits altered Na affinity and decreased thermal stability

The γ subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of γ in a native tissue environment, mice lacking a functional γ subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that γ is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2–/– dams. In adult Fxyd2–/– mouse kidney, splice variants of γ that have different nephron segment-specific expression patterns were absent. Purified γ-deficient renal Na,K-ATPase displayed higher apparent affinity for Na+ without significant change in apparent affinity for K+. Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na+ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that γ and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the γ subunit had increased thermal lability. Combined with other evidence that γ participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.

Functional Significance of Gap Junctional Coupling in Preimplantation Development

Abstract Gap junctional intercellular coupling allows cells to share low molecular weight metabolites and second messengers, thus facilitating homeostatic and developmental processes. Gap junctions make their appearance very early in rodent development, during compaction in the eight-cell stage. Surprisingly, preimplantation mouse embryos lacking the gap junction protein connexin 43 develop normally and establish full-term pregnancies despite severely reduced gap junctional coupling. It was suggested that this might be explained by the presence of at least five additional connexins known to be expressed in blastocysts. In the present study, we set out to clarify the number of connexins present in preimplantation rodent embryos and the role of gap junctional coupling, if any, in blastocyst development. We provide evidence from reverse transcription-polymerase chain reaction analysis that the genes encoding 3 additional connexins (connexin 30 or β6, connexin 36 or α9, and connexin 57 or α10) are also transcribed in preimplantation mouse embryos. Furthermore, we show that multiple connexins are expressed in rat preimplantation embryos, indicating that multiplicity of connexin expression may be a common feature of early mammalian embryogenesis. We could detect no up-regulation of any of 3 coexpressed connexins examined in mouse embryos lacking connexin 43. Impaired intercellular coupling caused either by the loss of connexin 43 or by treatment of cultured embryos with the gap junctional coupling blocker 18α-glycyrrhetinic acid (AGA) had no discernable effect on either apoptosis or glucose utilization, parameters known to be affected by gap junctional coupling in other contexts. These results, taken together with the reported inability of AGA to perturb blastocyst formation, imply that gap junctional coupling is not essential during this developmental period. We propose that connexin expression and the assembly of multiple types of gap junction channels in preimplantation embryos facilitates the diversification of communication pathways that will appear during postimplantation development. New evidence of this diversification is presented using rat blastocyst outgrowths.

Expression of Pannexin Isoforms in the Systemic Murine Arterial Network

Aims: Pannexins (Panx) form ATP release channels and it has been proposed that they play an important role in the regulation of vascular tone. However, distribution of Panx across the arterial vasculature is not documented. Methods: We tested antibodies against Panx1, Panx2 and Panx3 on human embryonic kidney cells (which do not endogenously express Panx proteins) transfected with plasmids encoding each Panx isoform and Panx1–/– mice. Each of the Panx antibodies was found to be specific and was tested on isolated arteries using immunocytochemistry. Results: We demonstrated that Panx1 is the primary isoform detected in the arterial network. In large arteries, Panx1 is primarily in endothelial cells, whereas in small arteries and arterioles it localizes primarily to the smooth muscle cells. Panx1 was the predominant isoform expressed in coronary arteries, except in arteries less than 100 µm where Panx3 became detectable. Only Panx3 was expressed in the juxtaglomerular apparatus and cortical arterioles. The pulmonary artery and alveoli had expression of all 3 Panx isoforms. No Panx isoforms were detected at the myoendothelial junctions. Conclusion: We conclude that the specific localized expression of Panx channels throughout the vasculature points towards an important role for these channels in regulating the release of ATP throughout the arterial network.

The control of male fertility by 1,2,3-trihydroxypropane (THP; glycerol): Rapid arrest of spermatogenesis without altering libido, accessory organs, gonadal steroidogenesis, and serum testosterone, LH and FSH

This study examines the effects of intratesticular injection of aqueous 1,2,3-trihydroxypropane (THP; glycerol) solution on male reproductive biology. In a series of experiments, Sprague-Dawley rats of various ages (48-101 days) were injected with 50-200 microliters per testis and various parameters were studied for up to 21 weeks later. While an injection of THP resulted in testicular weight reduction of 45-60% within 2 weeks, the weights of prostate and seminal vesicles were not affected for the duration of the experiments. The number of sperm per epididymis in the THP-treated rats declined rapidly and was reduced by 99.99% (of controls) after the 3rd mating. THP-treated males mated with virgin females at the same frequency as control rats but all were infertile after the 3rd mating and remained infertile for the duration of the tests (21 weeks after treatment). In vitro studies showed that metabolism of 14C-progesterone by testicular homogenates was not altered quantitatively or qualitatively by THP treatment. Serum levels of androgens, LH and FSH of THP-treated rats did not differ significantly from the controls. Histologically and histochemically, the Leydig cells appeared to be normal, but the seminiferous tubules of THP-treated testes were devoid of spermatogenic cells within 2 weeks of a single treatment. It is concluded that direct injection of THP acts as a potent inhibitor of spermatogenesis resulting in long-term infertility without affecting steroidogenesis, libido, secondary sex characteristics, mating behaviour or serum hormone levels.

Effect of prenatal and neonatal exposure to lead on gonadotropin receptors and steroidogenesis in rat ovaries

Sprague-Dawley rats were treated with lead chloride (20 or 200 ppm) or sodium chloride (controls) in their drinking water, either prior to pregnancy or during pregnancy and lactation, and female offspring were examined at weaning (21 d) or at 150 d. Other female rats were treated from d 21 to 35. Tissue (blood, kidney, bone) lead levels, body, ovary, and uterus weights, ovarian steroidogenesis, and gonadotropin (luteinizing hormone and follicle-stimulating hormone) levels, and gonadotropin-receptor binding were determined. Prenatal and/or postnatal exposure to lead at these levels (20 and 200 ppm) did not affect tissue weights but did cause a significant decrease in gonadotropin-receptor binding in the prepubertal, pubertal and adult females. Conversion of progesterone to androstenedione and dihydrotestosterone was significantly decreased in 21-d-old rats; in 150-d-old females, the prenatal and/or postnatal exposure to lead resulted in significantly increased conversion to the 5-alpha-reduced steroids, normally high during puberty. The results demonstrate that lead exposure prior to mating may affect gonadotropin-receptor binding in the offspring and that lead exposure (in utero, via mothers milk, or post weaning) may significantly alter steroid production and gonadotropin binding in ovaries of the prepubertal, pubertal, and adult female.

Contributions of Na+/H+ exchanger isoforms to preimplantation development of the mouse

Previous work provided evidence of Na+/H+ exchanger activity in the apical domain of mouse trophectodermal plasma membranes that provides a route for entry of extracellular Na+ (Manejwala et al., 1989). This activity was hypothesized to contribute to the trans‐trophectodermal Na+ flux that is required for blastocoel expansion. In the present work, we have used reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunocytochemistry to identify members of the Na+/H+ exchanger (NHE) family that are likely to participate in this process. When cDNA preparations from ovulated oocytes and several stages of preimplantation development were tested with PCR primers specific for the NHE‐1, ‐2, ‐3, and ‐4 isoforms of the exchanger, only amplicons representing the NHE‐1 and NHE‐3 isoforms were detected. The identity of these amplicons was confirmed by direct sequencing. NHE‐1 mRNA is present in oocytes and in all preimplantation stages, increasing threefold on a per embryo basis between the 4‐cell and blastocyst stages. NHE‐3 mRNA, on the other hand, was only detected in oocytes. Immunocytochemical analysis of blastocysts revealed that NHE‐1 is localized in the basolateral domain of the trophectoderm, whereas NHE‐3 is localized in the apical domain, a situation like that in epithelia of adult organs. We conclude that NHE‐3, an oogenetic product that persists into the blastocyst stage, is the Na+/H+ exchanger isoform most likely to be involved in blastocoel expansion. Mol. Reprod. Dev. 50:146–153, 1998.

Sustained azoospermia in squirrel monkey, Saimiri sciureus, resulting from a single intratesticular glycerol injection

Previous studies in rats had shown that a single intratesticular injection of glycerol resulted in long-term suppression of spermatogenesis without marked alterations in hormone levels. Studies were undertaken to determine the effect of similar treatment in squirrel monkeys (Saimiri sciureus). Ten monkeys received an intratesticular injection of saline (controls) and ten of glycerol solution (treated). Semen and blood samples were obtained on a weekly or bi-weekly basis one month prior to, during the 8 months following and at 22 months after the injection. Sperm numbers in the semen samples of controls remained at 160-435 x 10(6) per ml throughout the experiment. Sperm numbers in treated animals declined to near zero within two months and remained at zero. Serum testosterone and progesterone levels were not significantly different between control and treated animals. Serum LH and FSH levels were not significantly different between control and treated animals except during months 6-8 after the injection, when levels in the treated were higher. At termination (22 months), the weights and sperm contents of epididymides of the glycerol-treated animals were highly significantly reduced. Steroidogenesis (based on amounts and kinds of steroids formed from 14C-progesterone) by testicular tissue was not altered by the glycerol treatment when measured on a per testis basis. This is the first evidence that a single intratesticular injection of glycerol results in long-term suppression of spermatogenesis in primates, without altering testicular steroidogenesis and serum hormone levels.