Connexin 43 (Cx43) is the most abundant and ubiquitously distributed gap junction protein in testicular cells. Lack of Cx43 expression results in male infertility. We investigated whether Cx43 is expressed and regulated in Leydig, Sertoli and germinal cells at different stages of postnatal development. Cx43 was detected using three different antibodies shown by immunoblotting to be highly specific. At different postnatal ages Cx43 localization was compared in serial or double labeled testicular cryosections with immunocytochemical distribution of steroidogenic enzyme, 3 ␤hidroxysteroid-dehydrogenase (3␤HSD), Mullerian inhibitory hormone (MIH), and germinal nuclear cell antigen (GNCA1), which are specific markers of interstitial Leydig, Sertoli and germinal cells, respectively. In the interstitium, round cell clumps (RCC) with lipid droplets positive for 3␤HSD and Cx43 were frequently found at intertubular areas at birth and Cx43 was mainly localized at cell membrane appositions. From day 3, the number and size of 3␤HSD-positive RCC started to decrease, and reached a minimum at 7-14 dpp; Cx43 expressed by them is progressively downregulated. From day 21 an increase in the size and number of RCC positive for Cx43 and 3␤HSD was found that continued at 24, 26 and 28 days and reached a maximum at 35 and 60 dpp. Biphasic expression of interstitial Cx43 and 3␤HSD was also found to be positively and temporally correlated with fluctuations in intratesticular testosterone content at all ages studied. In the seminiferous cord (SC), Cx43 was expressed at birth between adjacent Sertoli cells (MIH positive) localized at the periphery, as well as in their cytoplasm projections that surround centrally localized gonocytes. From days 3 to 7, Cx43 labeling increased in Sertoli cells mainly at their apical border. At day 14, Cx43 distribution in Sertoli cells changed from apical to basal in parallel to migration of germinal (GNCA1-positive) cells from the periphery to the center of the SC. At all these ages, Cx43 was also localized at cell borders between Sertoli and germinal cells. In conclusion, this study demonstrates that Cx43 in Leydig cells is regulated during postnatal development in an age and functional dependent manner. In the tubule, it is demonstrated that Cx43 is modulated in Sertoli cells during the neonatal and prepubertal period. We also provide evidence for the first time that Cx43-gap junctions communicate between Sertoli and germinal cells before and during the first wave of spermatogenesis. Anat Rec 264: [13][14][15][16][17][18][19][20][21][22][23][24] 2001.

Developmental studies have shown that connexin43 (Cx43) is expressed in the ovary from the first day of life and throughout the rest of postnatal development. In both mouse embryonic ovaries and testes, target-directed deletion of Cx43 gene induces a significant decrease in germinal cells, but the exact mechanism determining this reduction remains unknown. Moreover, recently we found that Cx43 is abundantly expressed in mouse testes from the earliest stages of its fetal development. In the present work we investigate whether Cx43 transcript and protein are expressed in mouse embryonic ovaries. Total RNA was analyzed with specific Cx43 oligonucleotides in RT-PCR studies. A Cx43 PCR product was detected in ovaries at 16.5 and 18.5 days postcoitum (dpc). Bands of 43-45 kDa, characteristic of Cx43, were detected in immunoblots of total homogenates of ovaries at 14.5 and 18.5 dpc. Cell type-specific expression of Cx43 was investigated using double-labeled sections incubated with specific antibodies against Cx43 and the enzyme 3␤-hydroxysteroid dehydrogenase (3␤HSD) or a germ cell nuclear antigen (GCNA1), which are cell markers of steroidogenic and germinal cells, respectively. At 18.5 dpc, Cx43 was found in conglomerates of 3␤HSD-positive cells. Cx43 was also localized at homocellular junctions between parenchyma pregranulosa cells, and at heterocellular junctions between pregranulosa and germinal cells. At these two latter localizations, Cx43 was traced back to 12.5 dpc.In conclusion, this study demonstrates for the first time that from the earliest stages of embryonic ovary development, Cx43 is expressed in principal cell types involved in control of female fertility. These data suggest that the gap junctions formed with Cx43 between somatic and germinal cells may be necessary for prenatal expansion of germinal cells at initial stages of fetal gonadal development. Anat Rec Part A 271A: 360 -367, 2003.