Steroid hormones regulate cell function via specific receptors, members of a super family of ligand activated transcription factors, expressed in their target tissues. A second oestrogen receptor (ER beta) has recently been shown by RT-PCR to have a wide tissue distribution distinct from that of oestrogen receptor alpha (ER alpha). We have raised a polyclonal antiserum using a peptide specific for ER beta in order to determine the cellular sites of expression of the receptor. In the adult rat ER beta was localised to cell nuclei in a wide range of tissues including ovary, oviduct, uterus, lung, adrenal, seminal vesicle, bladder, heart, prostate and testis. In the ovary ER beta was present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea whereas ER alpha was undetectable in these cell types. In the uterus ER beta and ER alpha were both present in epithelial cells lining the lumen and glands. In the lung ER beta was present in the cells lining the bronchioles and alveoli as well as in smooth muscle. In bladder and seminal vesicle immunostaining was intense in epithelial cells but the receptor was also expressed in nuclei of smooth muscle cells. Cell nuclei of the heart ventricle were immunopositive for ER beta as were most cells of the adult rat adrenal. In the seminiferous epithelium of the testis, nuclei of Sertoli cells were immunopositive but expression was not stage dependent. In conclusion, immunohistochemistry has proved invaluable in visualising specific sites of expression of ER beta in complex tissues including those of the reproductive tract.

The aim of the present study was to identify specific, novel germ cell markers that could be used to monitor normal and abnormal spermatogenesis. Of several cloned cDNAs isolated from an adult rat testis cDNA library using an expression screening strategy, clone 813B4 (700 base pairs) hybridized exclusively to three mRNA transcripts in samples isolated from rat testes on and after Day 21 of life and to epididymides from some, but not all, adult rats. After further screening, two identical clones encoding a 2.2-kilobase cDNA (KTT4) were isolated and found to contain an open reading frame of 578 amino acids including two leucine zipper motifs. On Northern blots, KTT4 mRNA was abundant in samples from round spermatids, and homologous mRNAs were present in testes from mice and marmosets. A zoo blot revealed that the KTT4 gene is conserved in humans, monkeys, mice, dogs, and cattle. On sections of rat testes, KTT4 mRNA was first detectable in pachytene spermatocytes at stage VII and thereafter was abundant in round and elongating spermatids until step 15. Expression of KTT4 was not altered by ethane dimethane sulphonate-induced androgen withdrawal, but in rats treated 14 days previously with methoxyacetic acid, a marked reduction in KTT4 was noted associated with the depletion of round spermatids. In conclusion, the present study identified a conserved gene expressed in meiotic and post-meiotic germ cells; database searches have shown it to be homologous to recently published sequences for an outer dense fiber protein of the sperm tail (Odf2/Odf84).

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Information on the organization of the spermatogenic cycle of the common marmoset (Callíthrix jacchus), a small New World primate, is limited to a single histological report on the differentiation of spermatids. In the present study we have used non-radioactive in-situ hybridization with a cRNA probe directed against marmoset protamine 2, on fixed sections of marmoset and human testis to elucidate the organization of mature germ cells within the seminiferous epithelium. Specificity of the probe was checked on Northern blots; mP2 hybridized exclusively to mRNA in samples extracted from marmoset and human testis. In sections from human and marmoset testis, positive staining for mRNA was confined to round and elongating spermatids and in the human was reduced in samples from patients with incomplete spermatogenesis. In the human, P2 mRNA was present in groups of cells consistent with the presence of more than one stage of the spermatogenic cycle in transverse sections of individual tubules. In the marmoset, P2-positive cells were detected as a continuous ring of staining in the majority of sections of tubules whilst in others only a group(s) of cells was positive. We conclude that the arrangement of the spermatogenic wave in this New World primate may be intermediate between that seen in rodents (segmental) and in the human (helical).

Iron is required for the normal development of germ cells during spermatogenesis. Because these cells have no direct access to systemic iron, there exists a shuttle system involving production and secretion of the iron-transporting protein transferrin by the Sertoli cells. Previous reports using cultures of immature Sertoli cells exposed to adult germ cells, or in vivo studies involving germ cell depleted adult rat testes, concluded that production of transferrin by Sertoli cells is modulated by germ cell complement. In the present study we have used in situ hybridisation with cRNA probes directed against the 5' and 3' ends of transferrin mRNA to examine the pattern of expression of transferrin in the immature and adult rat testis. Adult rats were treated with ethane dimethane sulphonate or methoxyacetic acid (MAA) to manipulate their testosterone levels or germ cell complement respectively. Initial findings obtained using the 3' probe showed a decrease in transferrin mRNA associated with round spermatid depletion. However, these data were not confirmed by in situ hybridisation when the 5' probe was used. The specificity of the probes was examined using Northern blotting and the 3' probe was found to hybridise to the germ cell transcript for hemiferrin even under conditions of high stringency. Examination of immature and pubertal rat testes by in situ hybridisation using the 5' transferrin-specific probe found that as early as 14 days of age the level of expression of transferrin mRNA was clearly different between tubules, and the mRNA appeared to be expressed in Leydig cells on and after day 31. In the adult rat testis, maximal expression of transferrin mRNA was found at stages VIII-XIV, calling into question the interpretation of the results of some previous studies showing expression of transferrin mRNA at all stages of the spermatogenic cycle. This stage-specific pattern of expression was not altered by acute germ cell depletion using MAA. However, Northern blot analysis showed a statistically significant increase in transferrin mRNA expression at 7 days after MAA treatment when pachytene spermatocytes were depleted from tubules at all stages of the spermatogenic cycle at which transferrin is normally expressed. In conclusion, we found that transferrin mRNA expression was not modulated by round spermatids as has been reported previously but that meiotic germ cells may influence expression of transferrin at specific stages of the spermatogenic cycle.

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Several recent articles have reported localization of specific mRNAs in the rat testis to stage IX and X seminiferous tubules using in-situ hybridization. In all cases the expression was located basally in the tubules and appeared as discrete round clusters of grains close to the lamina propria. The localization was interpreted as being in Sertoli cells or leptotene spermatocytes. In this study we demonstrate that this pattern is most probably due to artefactual binding of probes to the residual body (RB). In the present study testicular tissue, perfusion-fixed with Bouin's and embedded in paraffin, was used, as this resulted in excellent morphological preservation such that RBs within tubules at stages VIII-X were clearly distinguishable. RNA content of the RBs was demonstrated at stages VIII-X using methyl green pyronin staining, and could be eliminated by pretreatment with RNAse or trichloroacetic acid. Localization of mRNAs for 11 seminiferous tubule proteins was assessed using 35S-labelled and digoxigenin-labelled riboprobes (activin receptor-II, alpha-inhibin, transferrin, androgen-binding protein (ABP), cyclic protein-2 (CP-2), CREM, sulphated glycoproteins 1 and 2 (SGP-1 and SGP-2), transition protein 2 (TP-2) and cystatin-C), and digoxigenin-labelled oligonucleotide probes (transition protein-1 (TP-1), TP-2 and protamine-1). All of these probes showed localization to the correct cell type(s) within the seminiferous epithelium. In addition, six antisense riboprobes (activin receptor-II, CREM, SGP-2, CP-2, cystatin C and alpha-inhibin) showed hybridization to basally located residual bodies in tubules at stages IX-X on one or more occasions, whereas residual bodies around the edge of the lumen (stage VIII) or in transit through the seminiferous epithelium showed no hybridization; sense probes showed no localization to residual bodies. A common feature of the probes which localized to the basal RBs was that they had been prepared using cDNA cloned into Bluescript SK- vector such that the antisense strand was generated from the T7 polymerase promotor. A cRNA prepared using T7 polymerase and Bluescript vector alone and a GC-rich 27mer oligonucleotide corresponding to the region of the multiple cloning site of Bluescript adjacent to the T7 site both localized uniquely to basal RB. It is concluded that the hybridization seen within RBs is probably a subtle artefact unique to RBs undergoing dissolution following fusion with Sertoli cell lysosomes, and may reflect nonspecific hybridization to GC-rich fragments of RNA.

Using testes fixed by perfusion with Bouin's fluid and embedded in paraffin wax, this study has established methods for combining in situ hybridization and immunocytochemistry on the same section to colocalize mRNA and protein for transition protein-1 (TP-1) and sulfated glycoprotein-1 (SGP-1), respectively. It was found that SGP-1 could be detected in tissue sections subsequent to the detection of TP-1 mRNA in situ. The finding that 1) the tissue pretreatments required to permeabilize the section and to allow access to the probe, and 2) the hybridization conditions themselves, had no adverse effect on the detection of antigen, eases the performance of this technique. On this basis, important information could be obtained on the transcriptional and translational activity of spermatogenic cells, if related probes and antibodies are utilized.